T24 antigen for immunodiagnosis of taenia solium cysticercosis

ABSTRACT

The present disclosure relates to T24 nucleic acid sequences, amino acid sequences, and antibodies. Methods for detecting and diagnosing  Taenia solium  infection in a subject using the T24 sequences and specific binding agents are also disclosed. The T24 sequences disclosed herein can be formulated into a pharmaceutical composition for administration to a subject. For example, the disclosed T24 polypeptides can also be administered to a subject to stimulate an immune response in the subject, thereby protecting the subject against  T. solium  infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/471,950 filed May 19, 2003, herein incorporated by reference in itsentirety.

FIELD

This application relates to purified Taenia solium T24 sequences anduses thereof in the diagnosis and prevention of cysticercosis andneurocysticercosis.

BACKGROUND

The disease cysticercosis, caused by the larval form of Taenia solium,is endemic in all regions of the world where humans and pigs live inclose contact. The lifecycle of T. solium begins when swine, theintermediate hosts, ingest tapeworm eggs excreted in the feces of atapeworm carrier. The larvae hatch from the eggs and invade most tissuesof the swine, giving rise to the disease cysticercosis.

When humans ingest raw or undercooked meat from cysticercotic swine,tapeworms, or taeniasis, develop. Patients with taeniasis may exhibitepigastric discomfort, nausea, irritability, diarrhea, and weight loss.In addition, proglottids, or individual segments of the tapeworm thatare self-contained hermaphroditic reproductive units, may obstruct theappendix, biliary duct, pancreatic duct.

Humans may also ingest T. solium eggs present in contaminated food andwater and become infected with the larval form. After T. solium eggs areingested, cysticerci may develop in the subcutaneous tissues, muscles,heart, lungs, liver, brain, and eye. Although small numbers of viablecysticerci may fail to produce symptoms in the infected host, death ofthe larvae stimulate a marked inflammatory reaction, fever, musclepains, and eosinophilia. If the larvae invade the central nervoussystem, a single cyst may cause disease. The host may developmeningoencephalitis, epileptic seizures, dementia and other neurologicor psychiatric manifestations, and death can result from acuteintracranial hypertension. The various manifestations of neurologicdysfunction caused by T. solium infection are collectively termedneurocysticercosis. T. solium neurocysticercosis has a current worldwidetoll of 50 million cases with 50,000 deaths each year. Althoughneurocysticercosis is rarely acquired in the United States, it is commonin Latin America, Asia, sub-Saharan Africa, and Eastern Europe. Due tothe increased travel and immigration from highly endemic areas,detection and treatment of T. solium related diseases are U.S. publichealth priorities.

Diagnosis of cysticercosis historically relied on histologicalidentification of the parasite by biopsy or autopsy. Although radiologicmethods such as computed tomography or nuclear magnetic resonanceimaging are useful in diagnosing neurocysticercosis, they are often tooexpensive or inaccessible in developing countries. In addition, althoughserological diagnostic tests are available to identify T. soliuminfection and diagnose neurocysticercosis, current immunoelectrotransferblot (EITB) assays utilize purified, naturally-occurring T. soliumlarval proteins, making the assay reagents expensive and difficult toproduce (see U.S. Pat. No. 5,354,660 to Tsang et al.).

In developing countries where T. solium-related diseases are endemic,access to diagnostic assays can be limited due to the high cost of usingT. solium antigens that are produced using complicated purificationprocedures. Furthermore, because cysticercosis is most prevalent inrural areas of developing countries, a field test is needed forepidemiological studies and surveillance. A field assay usinginexpensive and reliable reagents would be an important tool in breakingthe transmission cycle of the parasite, enabling the on-site diagnosisof infected pigs and immediate treatment with anti-helminthic agentssuch as oxfendazole. A field diagnosis of cysticercosis would also serveas an economic benefit to pig farmers, because uninfected pigs command ahigher price.

Therefore, there is a need to clone the T. solium glycoproteins so thatrecombinant proteins can be readily produced for field testing anddiagnosis of cysticercosis and neurocysticercosis.

SUMMARY

In order to develop a simple detection assay for field use, the T.solium T24 diagnostic protein was cloned and sequenced. Although themolecular weight of the protein was previously described in U.S. Pat.No. 5,354,660, its amino acid and nucleic acid sequences were notpreviously known. In addition, it was determined that several T. soliumproteins (about nine) co-migrate at this molecular weight, makingpurification and sequencing of T24 difficult. Cloning of T24 permitsrapid and inexpensive recombinant or synthetic production of T24protein, as compared to the more cumbersome process of isolating T24from T. solium. Recombinant or synthetic T24 proteins, alone or incombination with other T. solium larval proteins, can be used inimmunoassays for the detection of T. solium and diagnosis ofcysticercosis and neurocysticercosis.

The T24 protein is a membrane protein that migrates at approximately 24kilodaltons (kDa) using a non-reducing SDS-PAGE. T24 was previouslyreferred to in the literature as GP24; however, it is referred to hereinas T24 because the protein extracts into the detergent phase of TritonX-114. A T24 nucleic acid sequence is provided in SEQ ID NO: 1, and thecorresponding amino acid sequence in SEQ ID NO: 2. The coding sequenceis provided as nucleotides 33-707 of SEQ ID NO: 1. However, thisdisclosure is not limited to these particular sequences. Variants,fusions, and fragments of SEQ ID NOS: 1 and 2 are encompassed by thisdisclosure, as long as the protein retains immunogenic activity (orencodes a protein having immunogenic activity), such as amino acids104-195 of SEQ ID NO: 2 and nucleotides 342-617 of SEQ ID NO: 1.

The disclosed 124 sequences can be used to produce large quantities ofhighly pure T24 peptide or nucleic acid molecule, and can be used inimmunoassays for the detection of T. solium and diagnosis ofcysticercosis and neurocysticercosis. For example, T24 amino acidsequences can be used to synthesize T24 antigens or T24 antigenicfragments (such as amino acids 104-195 of SEQ ID NO: 2) using knownchemical synthesis techniques. In addition, nucleic acid moleculesencoding for T24 T. solium antigen can be used to recombinantly producethe T24 antigens and antigen fragments, and are also useful as molecularprobes or primers for the detection of ribonucleic acid (RNA) anddeoxyribonucleic acid (DNA) involved in transcription and translation ofT. solium peptides. T24 molecular probes and primers provide highlyspecific and sensitive means to detect and measure T. solium larvalpolypeptides in tissues and cells. Diagnostic and analytical methods andkits are provided for detection and measurement of T24 T. soliumantibodies, proteins, and nucleic acids in a variety of samples. Suchkits can be in any configuration known to those of ordinary skill in theart.

Recombinant or synthetic T. solium T24 polypeptides can be used indiagnostic kits to detect the presence and quantity of T. soliumantibodies, which are diagnostic or prognostic for the occurrence,recurrence or treatment of diseases such as cysticercosis andneurocysticercosis. Methods provided herein include immunoassays thatcan be used to detect or quantitate the presence of anti-T24 T. soliumantibodies in a sample, such as human or pig biological fluids ortissue. Such antibodies can also bind to naturally occurring T. solium flarval antigens, for instance naturally occurring antigens that havebeen isolated by lentil lectin affinity chromatography. In one example,an immunoassay includes one or more recombinant or synthetic T. soliumlarval peptides or antigenic fragments thereof, such as T24, for thedetection of anti-larval antibodies in a sample. An example of such animmunoassay is a rapid immunochromatographic diagnostic test (such as acard test) containing recombinant T24 peptides or antigenic fragmentsthereof, immunoreactive with anti-T24-T. solium antibodies in a sample.Other exemplary methods employ immunoblot and ELISA assays.

This disclosure provides simple, sensitive methods for the diagnosis ofcysticercosis or neurocysticercosis, and compositions for use in suchmethods. For example, methods are provided for detecting T. soliumcysticercosis, such as diagnosis or monitoring of T. solium infection inhumans and animals, which is inexpensive, sensitive, and accurate, withlittle or no cross-reactivity. Stable reagents that can be relativelyinexpensively produced are provided that allow for the detection of T.solium in a sample. For example, the reagents can include compositionscontaining recombinant or synthetic T. solium larval peptides such asT24. In specific examples, the results produced from the disclosedassays can be interpreted without the use of instrumentation or specialtemperature conditions.

Other methods allow one to detect the presence of T24 T. solium nucleicacids, using nucleic acid hybridization and amplification assays.Nucleic acid probes and primers can be generated based on the nucleicacid sequence shown in SEQ ID NO: 1, or fragments thereof such asnucleotides 342-617 of SEQ ID NO: 1. In one example, probes or primersinclude at least 10 contiguous nucleotides of SEQ ID NO: 1, for exampleat least 15, at least 20, at least 25, or even at least 50 contiguousnucleotides of SEQ ID NO: 1.

Compositions and methods for detecting T. solium infection anddiagnosing diseases related to T. solium n infection are provided. Insome examples, compositions include one or more purified recombinant orsynthetic immunogenic, or immunodominant, polypeptides or peptides (orfragments thereof) of the T. solium helminth larvae, for instance T24 orantigenic fragments thereof. In some examples, compositions include oneor more isolated T. solium larvae nucleic acid sequences, such as anisolated T24 nucleic acid sequence. In yet other examples, compositionsinclude one or more antibodies that recognize T. solium larvae antigens,such as an anti-T24 antibody.

Recombinant or synthetic T. solium T24 peptide can be administered to asubject, alone or in the presence of a pharmaceutical carrier oradjuvant, to generate an immune response against T24 in the subject,thereby protecting the subject against T. solium infection and reducingor preventing T. solium infection or related disease.

These and other features and advantages of the present disclosure willbecome apparent after a review of the following detailed description andexamples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a digital image showing the results from T. solium cystshomogenized and extracted with Triton X-114. Lane 1 contains molecularweight markers. Lane 2 is a silver stain of proteins in the aqueousphase. Lane 3 is a silver stain of proteins in the detergent phase. Lane4 is a western blot of proteins in the detergent phase probed with acysticercosis positive sera pool. Three membrane proteins areidentified, GP50, T42, and T24.

FIGS. 2A and 2B are digital images showing the (A) antibody reactivityof T24 characterized with a panel of cysticercosis positive sera andcompared to (B) the antibody reactivity of the western blot GP24. In allcases, the antibody reactivities of the sera with the two antigens wereconcordant, demonstrating that M24 and GP24 are the same protein. Fromprotein sequencing data, it appears that T42 is a dimer of T24.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows a nucleic acid sequence of T24.

SEQ ID NO: 2 shows a protein sequence of T24, encoded by SEQ ID NO: 1.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. As used herein, thesingular forms “a” or “an” or “the” include plural references unless thecontext clearly dictates otherwise. For example, reference to “a T24protein” includes one or a plurality of such proteins and reference to“the T24 antibody” includes reference to one or more T24 antibodies andequivalents thereof known to those skilled in the art and so forth.

The term “or” refers to a single element of stated alternative elementsor a combination of two or more elements, unless the context clearlyindicates otherwise. For example, the phrase “a T24 nucleic acid or aT24 protein” refers to a T24 nucleic acid, a T24 protein, or acombination of a T24 nucleic acid and a T24 protein. As used herein,“comprises” means “includes.” Thus, “comprising a T24 peptide,” means“including a T24 peptide,” without excluding additional elements.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, such as pigs.

Antigen: An agent that can stimulate the production of antibodies or aT-cell response in a subject, such as compositions that are administeredto the subject. An antigen reacts with the products of specific humoralor cellular immunity, including those induced by heterologousimmunogens. The term “antigen” includes all related antigenic epitopes.Antigenic determinant refers to a region of a protein recognized by anantibody. In one example, an antigen is a T24 antigen that induces animmune response, such as the production of anti-T24 antibodies, in asubject.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA can be synthesized by reverse transcription frommessenger RNA extracted from cells.

Conservative substitution: One or more amino acid substitutions, such as2, 3, 5, 10, or 20 substitutions, for amino acid residues having similarbiochemical properties. In one example, 2-10 conservative substitutionsare included in a peptide, such as 2-5 or 4-9 conservative substitutionsin a peptide. Typically, conservative substitutions have little to noimpact on the activity of a resulting polypeptide. For example, aconservative substitution is an amino acid substitution in a peptidethat does not substantially affect the function of the peptide. In aparticular example, a conservative substitution is an amino acidsubstitution in a T24 peptide, such as a conservative substitution inSEQ ID NO: 2 or a peptide including amino acids 104-195 of SEQ ID NO: 2,which does not significantly decrease immunogenicity of T24.

Methods which can be used to determine the amount of immune responsegenerated by a variant T24 epitope are disclosed. For example, analanine scan can be used to identify which amino acid residues in a T24protein can tolerate an amino acid substitution. In one example, theimmune response generated is not decreased by more than 25%, for examplenot more than 20%, for example not more than 10%, when an alanine, orother conservative amino acid (such as those listed below), issubstituted for one or more native amino acids, as compared to an immuneresponse generated in the presence of a native T24 protein, such as SEQD NO: 2, or amino acids 104-195 of SEQ ID NO: 2.

A peptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpolypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. Alternatively, a peptide can beproduced to contain one or more conservative substitutions by usingstandard peptide synthesis methods.

Substitutional variants are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Examples of amino acids which can be substituted for anoriginal amino acid in a protein and which are regarded as conservativesubstitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glufor Asp; Ser for Cys; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Glnfor His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leuor Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyrfor Trp; Trp or Phe for Tyr; and Ile or Leu for Val. Further informationabout conservative substitutions can be found in, among other locationsin, Ben-Bassat et al., (J. Bacteriol. 169:751-7, 1987), O'Regan et al.,(Gene 77:237-51, 1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994),Hochuli et al., (Bio/Technology 6:1321-5, 1988) and in standardtextbooks of genetics and molecular biology.

Deletion: The removal of a sequence, such as a nucleic acid or aminoacid sequence, the regions on either side being joined together.

Degenerate variant: A polynucleotide encoding a peptide, such as T24,that includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequences areincluded as long as the amino acid sequence of the peptide, such as T24,encoded by the nucleotide sequence is unchanged. I Detection: Theability to quantitatively or qualitatively determine the presence of abiomolecule under investigation. In one example the biomolecule is oneor more T. solium larval proteins, such as T24, GP50, GP39-42, GP21,GP18, GP14 or GP13.

Enhance: To improve the quality, amount, or strength of something. Inone example, a therapy enhances the immune system if the immune systemis more effective at reducing infection by T. solium or reducing thedevelopment of one or more symptoms associated with cysticercosis orneurocysticercosis. In a particular example, a T24 epitope reduces asubject's susceptibility to a T. solium infection. Such reduction can bemeasured using any bioassay known in the art, for example, an ELISAassay.

Functionally Equivalent: A functionally equivalent molecule is amolecule that is altered from the original molecule, but retainsessentially the same functions as the non-altered molecule. In oneexample, a functionally equivalent protein includes one or more aminoacid alterations and retains a function of the unaltered protein, suchas it specifically binds an antibody that binds an unaltered form of theeptiope, or wherein the epitope with one or more sequence alterationsretains the ability to induce an immune response in a subject.

Examples of sequence alterations include, but are not limited to,conservative substitutions, deletions, mutations, frameshifts, andinsertions. In one example, a given polypeptide binds an antibody, and afunctional equivalent is a polypeptide that binds the same antibody.Thus a functional equivalent includes peptides which have the samebinding specificity as a polypeptide, and which can be used as a reagentin place of the polypeptide (such as in a diagnostic assay or vaccine).In one example a functional equivalent includes a polypeptide whereinthe binding sequence is discontinuous, wherein the antibody binds alinear epitope. Thus, if the peptide sequence is MALSCGGDFL (amino acids1-10 of SEQ ID NO: 2) a functional equivalent includes discontinuousepitopes, which may can appear as follows (**=any number of interveningamino acids): NH2-**-M**A**L**S**C**G**G**D**F**L-COOH. This polypeptideis functionally equivalent to SEQ ID NO: 2 if the three dimensionalstructure of the polypeptide is such that it can bind a monoclonalantibody that binds SEQ ID NO: 2, or if it retains the ability to bestimulate an immune response in a subject.

Immune response: A change in immunity, for example, a response of a cellof the immune system, such as a B cell, T cell, or monocyte, to astimulus. In one example, the response is specific for a particularantigen (an “antigen-specific response”), such as a response to a T24peptide antigen. In one example, an immune response is a T cellresponse, such as a CD4⁺ response or a CD8⁺ response. In anotherexample, the response is a B cell response, and results in theproduction of specific antibodies, for example antibodies that recognizeT24. In a particular example, an increased or enhanced immune responseis an increase in the ability of a subject to fight off a disease, suchas cysticerosis or neurocysticerosis.

Immune stimulatory composition: A pharmaceutical composition whichincludes a T24 protein (or a variant, fusion, or fragment thereof, suchas amino acids 104-195 of SEQ ID NO: 2), which when administered to asubject, results in the subject producing antibodies against a T24protein. The subject's response results in treatment of the subjectsuffering from cysticerosis or neurocysticerosis. Such compositions cancontain other molecules, for example a pharmaceutically acceptablecarrier or an adjuvant.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acidmolecules and proteins which have been “isolated” include nucleic acidmolecules and proteins purified by standard purification methods. Theterm also includes nucleic acid molecules and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acid molecules and peptides.

Modulating an Immune Response: The ability to increase or decrease animmune response in a subject, such as the ability to stimulate a CTLimmune response, by a desired amount. Agents that modulate an immuneresponse include, but are not limited to: T24 polypeptides (such as SEQID NO: 2 as well as fragments, variants, fusions, and polymorphismsthereof, for example amino acids 104-195 of SEQ ID NO: 2), T24 nucleicacid molecules encoding T24 peptides (such as SEQ ID NO: 1 as well asfragments, variants, fusions, and polymorphisms thereof, for examplenucleotides 342-617 of SEQ ID NO: 1), T24 specific binding agents, T24antisense molecules, and immunoreactive sensitized T cells sensitizedwith T24.

Nucleic acid molecule: A deoxyribonucleotide or ribonucleotide polymerin either single or double stranded form. Includes analogues of naturalnucleotides that hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides.

Oligonucleotide: A linear polynucleotide (such as DNA or RNA) sequenceof at least 9 nucleotides, for example at least 10, at least 12, atleast 15, at least 18, at least 24, at least 25, at least 27, at least30, at least 50, at least 100 or even at least 200 nucleotides long. Ina particular example, an oligonucleotide includes at least 9 contiguousnucleotides of SEQ ID NO: 1, such as at least 12, at least 15, at least18, at least 24, at least 25, at least 27, at least 30, at least 50, atleast 100 or even at least 200 contiguous nucleotides of SEQ ID NO: 1.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Peptide Modifications: One or more alterations to a native peptide. Thepresent disclosure includes T24 proteins, as well as analogues(non-peptide organic molecules), derivatives (chemically functionalizedpeptide molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of T24 that can generate an immuneresponse. T24 peptides having one or more modifications can be utilizedin the methods described herein. The peptides disclosed herein include asequence of amino acids, which may be either L- or D-amino acids,naturally occurring and otherwise.

Peptides can be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into a T24 peptide to selectand provide conformational constraints to the structure that result inenhanced stability.

Peptidomimetic and organomimetic embodiments are also within the scopeof the present disclosure, whereby the three-dimensional arrangement ofthe chemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid side chains, resulting in such peptido- and organomimetics ofthe proteins of this disclosure having measurable or enhanced ability tobind an antibody. For computer modeling applications, a pharmacophore isan idealized, three-dimensional definition of the structuralrequirements for biological activity. Peptido- and organomimetics can bedesigned to fit each pharmacophore with current computer modelingsoftware (using computer assisted drug design or CADD). See Walters,“Computer-Assisted Modeling of Drugs”, in Klegerman & Groves, eds.,1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove,Ill., pp. 165-174 and Principles of Pharmacology Munson (ed.) 1995, Ch.102, for descriptions of techniques used in CADD. Also included withinthe scope of the disclosure are mimetics prepared using such techniques.In one example, a mimetic mimics the immune response generated by a T24peptide or antigenic fragment thereof.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful herein are conventional. Remington's PharmaceuticalSciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15thEdition (1975), describes compositions and formulations suitable forpharmaceutical delivery of the peptides and nucleic acids hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as a powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: A linear nucleic acid sequence of at least 3nucleotides. Therefore, a polynucleotide includes molecules which are atleast 15, at least 20, at least 24, at least 27, at least 30, at least50, at least 100, at least 200, at least 500, at least 1000, or at least5000 nucleotides in length, and also nucleotides as long as a fulllength cDNA. A T24 polynucleotide encodes a T24 peptide.

In a particular example, a polynucleotide includes at least 9 contiguousnucleotides of SEQ ID NO: 1, such as at least 12, at least 20, at least30, at least 50, at least 100, at least 200, at least 250, at least 300,at least 400, at least 600 or even at least 800 contiguous nucleotidesof SEQ ID NO: 1.

Polypeptide: Any chain of amino acids at least six amino acids inlength, such as at least 10 amino acids, at least 14 amino acids, atleast 20 amino acids, at least 92 amino acids, even at least 100 aminoacids regardless of post-translational modification (such asglycosylation or phosphorylation). In one example, a polypeptide is aT24 peptide, such as SEQ ID NO: 2 or fragments thereof such as aminoacids 104-195 of SEQ ID NO: 2.

In a particular example, a polynucleotide includes at least 10contiguous amino acids of SEQ ID NO: 2, such as at least 12, at least20, at least 30, at least 50, at least 75, at least 90, at least 92, atleast 100, at least 150, at least 200 or even at least 210 contiguousamino acids of SEQ ID NO: 2.

Preventing or treating a disease: “Preventing” a disease refers toinhibiting the full development of a disease, for example preventingdevelopment or progression of cysticerosis or neurocysticerosis.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition related tocysticerosis or neurocysticerosis, such as inflammation, fever, musclepains, eosinophilia, meningoencephalitis, epileptic seizures, dementiaor other neurologic or psychiatric manifestations, or acute intracranialhypertension.

Probes and primers: Nucleic acid probes and primers can readily beprepared based on the T24 sequences provided herein.

A probe includes an isolated nucleic acid attached to a detectable labelor reporter molecule. Typical labels include, but are not limited to,radioactive isotopes, ligands, fluorophores, chemiluminescent agents,and enzymes. Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, for example, in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989) and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-lntersciences(1987).

Primers are short nucleic acids, such as DNA oligonucleotides about atleast 12 nucleotides in length. Primers can be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, for example by PCRor other nucleic-acid amplification methods known in the art. A “primerpair” refers to two primers, one having a forward designation and theother having a reverse designation (relative to their respectiveorientations on a double-stranded DNA molecule that includes a sense andantisense sequence), such that under amplification conditions theforward primer anneals to and primes amplification of the sense sequenceand the reverse primer anneal& to and primes amplification of theantisense sequence.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (Molecular Cloning. A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989), Ausubel et al., 1987, andInnis et al., PCR Protocols, A Guide to Methods and Applications, 1990,Innis et al. (eds.), 21-27, Academic Press; Inc., San Diego, Calif. PCRprimer pairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as Primer (Version 0.5,© 1991, Whitehead Institute for Biomedical Research, Cambridge, Ma.).

One of skill in the art will appreciate that the specificity of aparticular probe or primer increases with its length. Thus, for example,a primer consisting of 20 consecutive nucleotides of SEQ ID NO: 1 willanneal to a target sequence, such as a T24 homolog contained within agenomic DNA library, with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, to obtain greater specificity,probes and primers can be selected that include at least 25, at least30, at least 35, at least 40, at least 50 or more consecutivenucleotides of SEQ ID NO: 1.

In addition, T24 cDNA and gene sequences can be apportioned into halvesor quarters based on sequence length, and the isolated nucleic acidmolecules can be derived from the first or second halves of themolecules, or any of the four quarters. In particular, the DNA sequencesmay code for a unique portion of T24, such as an immunogenic fragment ofT24, such as amino acids 104-195 of SEQ ID NO: 2.

Promoter: An array of nucleic acid control sequences that directstranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of a polymerase II type promoter, a TATA element. A promoter alsooptionally includes distal enhancer or repressor elements which can belocated as much as several thousand base pairs from the start site oftranscription. Both constitutive and inducible promoters are included(Bitter et al., Meth. Enzymol. 153:516-44, 1987). Exemplary promotersare provided below.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its generative environment, for instancewithin a cell or in a biochemical reaction chamber. Therefore, apurified peptide is substantially separated from cellular components(such as nucleic acids, lipids, carbohydrates, and other polypeptides)that can accompany it. In another example, a purified peptidepreparation is one in which the peptide is substantially-free fromcontaminants, such as those that can be present following chemicalsynthesis of the peptide.

A preparation of substantially pure peptide, such as T24 peptide, can bepurified such that the desired peptide represents at least 50% by weightof a sample. In certain examples, a substantially pure peptiderepresents at least 60%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% or more by weight of the totalpeptide content of the preparation.

Examples of methods that can be used to purify a peptide, include, butare not limited to the methods disclosed in Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989, Ch.17). Protein purity can be determined by, for example, polyacrylamidegel electrophoresis of a protein sample, followed by visualization of asingle polypeptide band upon staining the polyacrylamide gel;high-pressure liquid chromatography; sequencing; or other conventionalmethods.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesis orby the artificial manipulation of isolated segments of nucleic acids,such as by genetic engineering techniques. Similarly, a recombinantprotein is one encoded for by a recombinant nucleic acid molecule, or aprotein produced by other synthetic methods using technology known inthe art.

Sample: Material obtained from a subject. In some examples, the sampleis a cell(s) obtained from a subject that includes genomic DNA, cDNA,RNA, or protein. Exemplary samples include, but are not limited to:peripheral blood, urine, saliva, tissue biopsy, surgical specimen, fineneedle aspriates, amniocentesis samples and autopsy material.

Sequence identity: The similarity between nucleic acid or amino acidsequences is expressed in terms of the similarity between the sequences.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or variants of a protein or nucleic acidmolecule disclosed herein, such as SEQ ID NOS: 1-2, will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:2374, 1988; Higgins andSharp, CABIOS 5:151-3, 1989; Corpet et al., Nucl. Acids Res.16:10881-90, 1988; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988; and Altschul et al., Nature Genet. 6:119-29, 1994.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the Nation Center for Biotechnology Information NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Variants of a peptide, such as a T24 peptide shown in SEQ ID NO: 2, oramino acids 104-195 of SEQ ID NO: 2 are typically characterized bypossession of at least 85% sequence identity counted over the fulllength alignment with the amino acid sequence shown in SEQ ID NO: 2 oramino acids 104-195 of SEQ ID NO: 2 using the NCBI Blast 2.0, gappedblastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment is performed using the Blast 2 sequences function, employingthe PAM30 matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 90%, at least 95%, at least 98%, or evenat least 99% sequence identity. When less than the entire sequence isbeing compared for sequence identity, homologs and variants willtypically possess at least 85% sequence identity over short windows of10-20 amino acids, and may possess sequence identities of at least 90%,at least 95%, or at least 98% depending on their similarity to thereference sequence. Methods for determining sequence identity over suchshort windows are described at the website that is maintained by theNational Center for Biotechnology Information in Bethesda, Md. One ofskill in the art will appreciate that these sequence identity ranges areprovided for guidance only; it is entirely possible that stronglysignificant homologs could be obtained that fall outside of the rangesprovided.

Similar methods can be used to determine the sequence identity betweentwo or more nucleic acid molecules. To compare two nucleic acidsequences, the BLASTN options can be set as follows: -i is set to a filecontaining the first nucleic acid sequence to be compared (such asC:\seq1.txt); -j is set to a file containing the second nucleic acidsequence to be compared (such as C:\seq2.txt); -p is set to blastn; -ois set to any desired file name (such as C:\output.txt); -q is set to-1; -r is set to 2; and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison between two sequences: C:B12seq-ic:\seq1.txt-j c:\seq2.txt-p blastn-o c:\output.txt-q-1-r2.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (1166+−1554*100=75.0).The percent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(15÷20*100=75). 1                  20 Target Sequence:ATGGGTCTCTCATGCGGCGG | || ||| |||| |||| | Identified Sequence:ACGGTTCTATCATCCGGCAG

The T24 nucleic acid molecules disclosed herein, such as SEQ ID NO: 1,include nucleic acids having at least 85%, at least 90%, at least 95%,at least 98%, or at least 99% identical to the nucleotide sequence ofSEQ ID NO: I or nucleotides 342-617 of SEQ ID NO: 1. In particularexamples, a nucleic acid molecule is substantially similar to thenucleotide sequence of SEQ ID NO: 1 or nucleotides 342617 of SEQ IDNO: 1. A first nucleic acid molecule is “substantially similar” to asecond nucleic acid molecule if, when the first nucleic acid molecule isoptimally aligned (with appropriate nucleotide deletions or gapinsertions) with the second nucleic acid molecule (or its complementarystrand) and there is nucleotide sequence identity of at least about 90%,for example at least about 95%, at least 98% or at least 99% identity.An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus, a T24 specific binding agent is an agent thatbinds substantially to a T24 polypeptide. In one example, the specificbinding agent is a monoclonal or polyclonal antibody. Methods forproducing antibodies are provided below.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bindto a specified protein are specific binding agents. These antibodyfragments include: (1) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule produced by digestionof whole antibody with the enzyme papain to yield an intact light chainand a portion of one heavy chain; (2) Fab′, the fragment of an antibodymolecule obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab)2, the fragment of the antibody obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; (4) F(ab′)2, adimer of two Fab′ fragments held together by two disulfide bonds; (5)Fv, a genetically engineered fragment containing the variable region ofthe light chain and the variable region of the heavy chain expressed astwo chains; and (6) single chain antibody (“SCA”), a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. Methods of makingthese fragments are routine. For example, construction of Fab expressionlibraries permits the rapid and easy identification of monoclonal Fabfragments with the desired specificity for a T24 protein.

T24 cDNA: As used herein, a T24 cDNA includes T24 cDNAs from anyorganism, such as T. solium. In one example, a T24 cDNA includes SEQ IDNO: 1, as well as fragments, fusions, and variants thereof. In someexamples, such fragments, variants, or fusions encode a T24 protein thatcan stimulate an immune response, encode a T24 protein that canimmunoreact with T24 antibodies, or combinations thereof. Exemplaryfragments include nucleotides 33-710 of SEQ ID NO: 1 or nucleotides342-617 of SEQ ID NO: 1. T24 cDNA can be derived by reversetranscription from the mRNA encoded by a T24 gene and lacks internalnon-coding segments and transcription regulatory sequences present in aT24 gene.

T24 gene: A gene that encodes a T24 protein from any organism, such asT. solium. A T24 gene includes the various sequence polymorphisms andallelic variants that exist within and between species.

T24 peptide: A protein encoded by a T24 gene or cDNA from any organism,such as T. solium. The immunogenic T24 larval T. solium membrane proteinmigrates at approximately 24 kDa on a non-reducing SDS-PAGE, andextracts into the detergent phase of Triton X-114. A T24 proteinincludes a full-length transcript, such as SEQ ID NO: 2, as well asfragments, fusions, and variants of SEQ ID NO: 2 that retain the abilityto stimulate an immune response, to immunoreact with T24 antibodies, orcombinations thereof. An exemplary fragment includes amino acids 104-195of SEQ ID NO: 2.

Therapeutically active molecule: An agent, such as a T24 protein ornucleic acid molecule, for example SEQ ID NOS: 1-2 (or variants,fusions, or fragments thereof), or T24 antibody, that can induce animmune response, as measured by clinical response (for example increasein a population of immune cells, or measurable reduction in T. soliuminfection). Therapeutically active agents can also include organic orother chemical compounds that mimic the effects of a T24 peptide, T24nucleic acid molecule, or T24 antibody.

Therapeutically Effective Amount: The preparations disclosed herein areadministered in therapeutically effective amounts. An effective amountis that amount of a pharmaceutical preparation that alone, or togetherwith further doses, stimulates the desired response.

In one example, a desired response is stimulation of a CTL response toT24, resulting in halting or slowing the progression of, or inducing aregression of a pathological condition or which is capable of relievingsigns or symptoms caused by the condition. One example of a therapeuticeffect is regression of one or more symptoms associated withcysticercosis or neurocysticercosis. Treatment can involve slowing theprogression of the disease temporarily, and can also include halting orreversing the progression of the disease permanently.

The therapeutically effective amount also includes a quantity of T24protein, nucleic acid molecule (such as SEQ ID NOS: 1-2, or variants,fusions or fragments thereof), or T24 antibody, sufficient to achieve adesired effect in a subject being treated. For instance, this can be theamount necessary to improve signs or symptoms a disease such ascysticercosis or neurocysticercosis, for example by decreasing one ormore of inflammation, fever, muscle pains, eosinophilia,meningoencephalitis, epileptic seizures, dementia or other neurologic orpsychiatric manifestations, and acute intracranial hypertension in asubject.

An effective amount of T24 protein can be administered in a single dose,or in several doses, for example daily, during a course of treatment.However, the effective amount can depend on the source applied (forexample T24 peptide isolated from a cellular extract versus a chemicallysynthesized and purified T24 peptide, or a variant or fragment that maynot retain full T24 activity), the subject being treated, the severityand type of the condition being treated, and the manner ofadministration. For example, a therapeutically effective amount of T24protein, can vary from about 0.01 mg/kg body weight to about 1 g/kg bodyweight, such as about 1 mg per subject.

The methods disclosed herein have equal application in medical andveterinary settings. Therefore, the general term “subject being treated”is understood to include all animals (such as humans and pigs) thatrequire an immune response against T24, for example to reduce symptomsassociated with cysticercosis or neurocysticercosis.

Transduced, transformed and transfected: A transduced, transformed, ortransfected cell is a cell into which an exogenous nucleic acid moleculehas been introduced, and wherein the nucleic acid molecule becomesstably replicated by the cell, either by incorporation of the nucleicacid molecule into the cellular genome, or by episomal replication. Avirus or vector ”transduces” or “transfects” a cell when it transfersnucleic acid molecules into the cell. These terms encompasses alltechniques by which a nucleic acid molecule can be introduced into sucha cell, including, but not limited to, transfection with viral vectors,transformation with plasmid vectors, and introduction of naked DNA byelectroporation, lipofection, and particle gun acceleration.

Transgene: An exogenous nucleic acid sequence, for example, a nucleicacid sequence supplied by a vector. In one example, a transgene encodesa T24 peptide.

Variants or fragments or fusion molecules: The disclosed T24 proteinsand nucleic acid molecules include variants, fragments, and fusionsthereof. DNA sequences which encode for T24 or fusion, fragment, orvariant thereof (for example a fragment or variant having at least 85%,at least 90%, or at least 95% sequence identity to nucleotides 33-710 ofSEQ ID NO: 1 or nucleotides 342-617 of SEQ ID NO: 1) can be engineeredto allow a T24 protein to be expressed in a host cells, such as abacteria, insect, mammalian, yeast, or plant cell. To obtain expression,the DNA sequence can be altered and operably linked to other regulatorysequences. The final product, which contains the regulatory sequencesand the therapeutic protein, is referred to as a vector. This vector canbe introduced into a host cell. Once inside the cell, the vector allowsthe protein to be produced.

A fusion protein including T24 (or variants, polymorphisms, mutants, orfragments thereof, such as amino acids 104-195 of SEQ ID NO: 2) linkedto other amino acid sequences that do not inhibit the desired activityof the protein, for example the ability to stimulate an immune response,interact with a T24 antibody, or both. In one example, the other aminoacid sequences are no more than 8, 9, 10, 12, 15, 20, 30, or 50 aminoacid residues in length.

One of ordinary skill in the art will appreciate that the DNA can bealtered in numerous ways without affecting the biological activity ofthe encoded protein. For example, PCR can be used to produce variationsin the DNA sequence which encodes a protein. Such variants can bevariants optimized for codon preference in a host cell used to expressthe protein, or other sequence changes that facilitate expression.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector can include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector can also include one or more therapeuticgenes or selectable marker genes and other genetic elements known in theart. A vector can transduce, transform or infect a cell, thereby causingthe cell to express nucleic acid molecules or proteins other than thosenative to the cell. A vector optionally includes materials to aid inachieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like. In one example, avector is a viral vector. Viral vectors include, but are not limited to,retroviral and adenoviral vectors.

Taenia solium T24 Polypeptides

Previously, several immunogenic T. solium peptides were identified bymolecular weight (see U.S. Pat. No. 5,354,660). However, until now theT24 protein and nucleic sequences had not been identified. Purificationand cloning of T24 was difficult because at least nine proteins,including T24, co-migrate at 24 kDa on a non-reducing SDS-PAGE.

Therefore, the T24 present in the band that migrates at 24 kDa on anon-reducing SDS-PAGE is not substantially pure. It is likely that theseprevious preparations of T24 were only about 30% pure by weight, due tothe contaminating proteins that co-migrate at 24 kDa.

Nucleic acid and protein sequences of T. solium T24 are disclosed.However, the disclosure is not limited to these particular sequences, asthe disclosed T24 sequences can be subject to various changes, such asinsertions, deletions, and substitutions. Such variations can providefor certain advantages, for example to increase biological activity.

In one example, substantially purified T24 protein is provided, forexample a T24 protein preparation that is at least 60% pure by weight,such as at least 80%, at least 90%, at least 95%, or even at least 98%pure by weight. A native T24 protein sequence is shown in SEQ ID NO: 2.However, this disclosure includes T24 protein variants, fusions, andfragments that can elicit or stimulate an immune response, react with aT24 antibody (such as those found in subjects having a T. soliuminfection), or combinations thereof. Variant sequences can contain asingle insertion, a single deletion, a single substitution, multipleinsertions, multiple deletions, multiple substitutions, or anycombination thereof (such as a single deletion together with multipleinsertions). One particular example of a T24 fragment includes aminoacids 104-195 of SEQ ID NO: 2. In one example, a T-cell that recognizesa T24 peptide can also recognize a variant of T24. In another oradditional example, such variant immunogenic peptide sequences canstimulate propagation of an immune cell (such as a T-cell). Thedisclosed T24 peptides are immunogenic, and can be used to elicit animmune response in a subject, such as those subjects having a T. soliuminfection.

Minor modifications of the primary amino acid sequence of T24 can resultin peptides which have substantially equivalent activity as compared tothe unmodified counterpart T24 peptide described herein. Suchmodifications may be deliberate, as by site-directed mutagenesis, or canbe spontaneous. All of the peptides produced by these modifications areincluded herein as long as an activity of variant peptide, such as theability to induce an immune response, still exists. In one example, suchvariants have at least 90% identity, such as at least 95% identity, tothe disclosed sequences. Particular examples of variants include thosehaving one or more, two or more, such as 2-10 conservative amino acidsubstitutions. In one example, a variant includes a single amino acidsubstitution, such as a single conservative amino acid substitution.

Purified T24 peptides can be at least 6 amino acids in length, such asat least 8, at least 9, at least 10, at least 11, at least 12, at least15, at least 20, at least 30, at least 50, at least 90, at least 92, oreven at least 100 amino acids in length. One skilled in the art willunderstand that fusion proteins including a T24 peptide can be evenlonger.

Certain compositions provided herein contain recombinant or synthetic T.solium T24 peptides that are immunoreactive with T. solium antibodies.T. solium antibodies are, in certain examples, derived from the sera,saliva, cerebrospinal fluid or urine of patients infected with T.solium. Alternatively, antibodies are produced in an experimental animalby administration of T24 peptide or an antigenic fragment thereof, suchas amino acids 104-195 of SEQ ID NO: 2. In specific examples, therecombinant or synthetic T24 peptides correspond to naturally occurringproteins having a molecular weight of approximately 24 kDa. In certaininstances, T24 peptides are encoded by a sequence that includes SEQ IDNO: 1, or variants, fragments, or fusions thereof, such as nucleotides33-710 of SEQ ID NO: 1 or nucleotides 342-617 of SEQ ID NO: 1.

The disclosed immunoreactive T24 polypeptides include 124 peptideanalogs, which include antigenic peptides containing amino acidsequences differing from those shown in SEQ ID NO: 2 (or amino acids104-195 of SEQ. ID NO: 2) by one or more amino acid substitutions at anyposition, or which have other molecules attached to amino acidfunctional groups within the sequence. Also disclosed are immunoreactivefragments (antigenic fragments) of T24 having substantially the sameantigenicity of T24 peptide, or the functional equivalent thereof. Incertain examples, T24 antigenic fragments contain amino acid sequencesthat are homologous or substantially homologous to T24. In a specificexample, a T24 antigenic fragment includes amino acid sequences that arehomologous or substantially homologous to SEQ ID NO: 2.

The T. solium T24 peptides described herein have a variety of uses. Forexample T24 peptides or antigenic fragments thereof can be used asreagents in immunoassays for the detection of T. solium antibodies asdescribed below. Furthermore, T. solium polypeptides can be employed inaffinity columns for isolating T. solium antibodies. Also, T24 peptidesthat bind to T. solium T24-antibodies with high specificity and aviditycan be labeled with a label or reporter group and employed forvisualization and quantitation in the assays described herein usingdetection techniques such as autoradiographic and membrane bindingtechniques. Such applications provide important diagnostic and researchtools. In addition, the disclosed T24 peptides can be used to stimulatean immune response, for example to prevent or treat cysticercosis orneurocysticercosis.

Production of Synthetic and Recombinant T24 T. solium Peptides

The T24 sequences provided herein can be used to produce T24 peptidesand T24 antigenic fragments using recombinant or synthetic methods knownto those skilled in the art.

Synthetic Methods

A synthetic T24 peptide refers to a 24 polypeptide formed in vitro byjoining amino acids in a particular order, for example using organicchemistry to form the peptide bonds. Methods for preparing syntheticpolypeptides are known in the art. For example, T24 peptides can besynthesized using solid phase techniques, cleaved from the resin, andpurified by preparative high performance liquid chromatography (forexample see Creighton, 1983, Proteins Structures And MolecularPrinciples, W. H. Freeman and Co., N.Y. pp. 50-60; Barany andMerrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis,Synthesis, Biology (Gross and Meienhofer (eds.), Academic Press, N.Y.,vol. 2, pp. 3-284 (1980)); Merrifield, et al., J. Am. Chem. Soc.85:2149-56 (1963); Stewart, et al., Solid Phase Peptide Synthesis (2nded., Pierce Chem. Co., Rockford, Ill. (1984)); S. B. H. Kent, BiomedicalPolymers, eds. Goldberg and Nakajima, Academic Press, New York:, pp.213-242, 1980; Mitchell et al., J. Org. Chem., 43:2845-52, 1978; Tam etal., Tet. Letters, 4033-6, 1979; Mojsov et al., J. Org. Chen:.,45:555-60, 1980; Tam et al., Tet. Letters, 2851-4, 1981; and Kent etal., Proceedings of the IV International Symposium on Methods of ProteinSequence Analysis, (Brookhaven Press, Brookhaven, N.Y, 1981); all ofwhich are hereby incorporated by reference). The composition of thesynthetic peptides can be confirmed by amino acid or sequence analysis(such as Edman degradation; see Creighton, 1983, Proteins, Structuresand Molecular Principles, W.H. Freeman and Co., N.Y., pp. 34-49).Smaller peptides can be joined to form larger polypeptides usingchemical ligation (Wilken and Kent, Current Opinion in Biotechnology4:412-26, 1998).

Briefly, solid phase synthesis is started from the C-terminal end of aT24 peptide by coupling a protected amino acid via its carboxyl group toa suitable solid support. The solid support used is not a criticalfeature, but ideally is capable of binding to the carboxyl group whileremaining substantially inert to the reagents utilized in the peptidesynthesis procedure. For example, a starting material can be prepared byattaching an amino-protected amino acid via a benzyl ester linkage to achloromethylated resin or a hydroxymethyl resin or via an amide bond toa benzhydrylamine (BHA) resin or p-methylbenzhydrylamine (MBHA) resin.Exemplary materials suitable for use as solid supports are well knownand include: halomethyl resins, such as chloromethyl resin orbromomethyl resin; hydroxymethyl resins; phenol resins, such as4-(a-[2,4-dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin;tert-alkyloxycarbonyl-hydrazidated resins, and the like. Such resins arecommercially available and their methods of preparation are known.

The acid form of T24 peptides can be prepared using a solid phasepeptide synthesis method using a benzyl ester resin as a solid support.The corresponding amides can be produced by using benzhydrylamine ormethylbenz-hydrylamine resin as the solid support. When BHA or MBHAresin is used, treatment with anhydrous hydrofluoric acid to cleave thepeptide from the solid support produces a peptide having a terminalamide group.

The α-amino group of each amino acid used in the synthesis can beprotected during the coupling reaction to prevent side reactionsinvolving the reactive α-amino function. Certain amino acids alsocontain reactive side-chain functional groups (such as sulfhydryl,amino, carboxyl, hydroxyl, etc.) that ideally are protected withappropriate protecting groups to prevent chemical reactions fromoccurring at those sites during peptide synthesis. Protecting groups arewell known.

Ideally, an α-amino protecting group will render the a-amino functioninert during the coupling reaction, will be readily removable aftercoupling under conditions that will not remove side chain protectinggroups, will not alter the structure of the peptide fragment, and willprevent racemization upon activation immediately prior to coupling.Similarly, side-chain protecting groups are chosen to render the sidechain functional group inert during the synthesis, are stable under theconditions used to remove the a-amino protecting group, and areremovable after completion of the peptide synthesis under conditionsthat will not alter the structure of the peptide.

Coupling of the amino acids can be accomplished by a variety of methods.Typical approaches involve either the conversion of the amino acid to aderivative that will render the carboxyl group more susceptible toreaction with the free N-terminal amino group of the peptide fragment,or use of a suitable coupling agent such as, for example,N,N′-dicyclohexylcarbodimide DCC) or N,N′-diisopropylcarbodiimide(DIPCDI). Hydroxybenzotriazole (HOBt) can be employed as a catalyst inthese coupling reactions. Appropriate synthesis chemistries aredisclosed in The Peptides: Analysis, Structure, Biology, Vol. 1: Methodsof Peptide Bond Formation (Gross and Meienhofer (eds.), Academic Press,N.Y., 1979); and Izumiya, et al., Synthesis of Peptides (MaruzenPublishing Co., Ltd., 1975).

Generally, synthesis of the peptide is commenced by first coupling theC-terminal amino acid, which is protected at the N-amino position by aprotecting group such as fluorenylmethyloxycarbonyl (Fmoc), to a solidsupport. Prior to coupling of Fmoc-Asn, the Fmoc residue has to beremoved from the polymer. Fmoc-Asn can, for example, be coupled to the4-(a-[2,4-dimethoxyphenyl]-Fmoc-amino-methyl)phenoxy resin usingN,N′-dicyclohexylcarbodimide (DCC) and hydroxybenzotriazole (HOBt) atabout 25° C. for about two hours with stirring. Following the couplingof the Fmoc-protected amino acid to the resin support, the α-aminoprotecting group is removed using 20% piperidine in DMF at roomtemperature.

After removal of the α-amino protecting group, the remainingFmoc-protected amino acids are coupled stepwise in the desired order.Appropriately protected amino acids are commercially available from anumber of suppliers (for example Novartis (Switzerland) or Bachem (CA)).As an alternative to the stepwise addition of individual amino acids,appropriately protected peptide fragments consisting of more than oneamino acid can also be coupled to the “growing” peptide. Selection of anappropriate coupling reagent, as explained above, is well known to thoseof skill in the art. If a T24 immunogenic peptide is relatively short inlength, this latter approach (the segment condensation method) is notthe most efficient method of peptide synthesis.

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in excess and the coupling is carried out in amedium of dimethylformamide (DMF), methylene chloride (CH₂Cl₂), ormixtures thereof. If coupling is incomplete, the coupling reaction canbe repeated before deprotection of the N-amino group and addition of thenext amino acid. Coupling efficiency can be monitored, for example usingthe ninhydrin reaction. Peptide synthesis reactions can be performedautomatically using a number of commercially available peptidesynthesizers (such as Biosearch 9500, Biosearch, San Raphael, Calif.).

The peptide can be cleaved and the protecting groups removed by stirringthe insoluble carrier or solid support in anhydrous, liquid hydrogenfluoride (HF) in the presence of anisole and dimethylsulfide at about 0°C. for about 20 to 90 minutes, in particular examples about 60 minutes;by bubbling hydrogen bromide (HBr) continuously through a 1 mg/10 mLsuspension of the resin in trifluoroacetic acid (TFA) for 60 to 360minutes at about room temperature, depending on the protecting groupsselected; or by incubating the solid support inside the reaction columnused for the solid phase synthesis with 90% trifluoroacetic acid, 5%water and 5% triethylsilane for about 30 to 60 minutes. Otherdeprotection methods can also be used.

The synthesized T24 peptides can be isolated and purified from thereaction mixture by means of peptide purification known to those ofskill in the art. For example, the peptides can be purified usingchromatographic procedures such as reverse phase HPLC, gel permeation,ion exchange, size exclusion, affinity, partition, or countercurrentdistribution.

Recombinant Methods

Recombinant T24 proteins can be produced by known methods. For example,a T24 nucleotide sequence can be inserted into a vector, such as aplasmid, and recombinantly expressed in a host cell to producerecombinant T24 peptides.

Briefly, a cloning vector, such as a plasmid or phage DNA is cleavedwith one or more restriction enzymes, and a T24 nucleic acid sequenceencoding a T24 protein (or variant or fragment thereof) is inserted intothe cleavage site and ligated. The cloning vector is introduced into ahost cell to produce the T24 peptide encoded by the nucleic acid.Suitable hosts include bacterial hosts, such as Escherichia coli andBacillus subtilis, as well as yeast, plant, insect, and mammalian celllines, and other cell cultures. Insect cell expression can be used togenerate a large amount of protein for use in a diagnostic assay, andcan be used when a protein includes disulfide bonds (such as T24).Exemplary insect cell lines include the D. melanogaster Schneider 2 (S2)cell line (American Type Culture Collection (ATCC) No. CRL-1963), D.melanogaster Kc1 cells, gypsy moth cell line, IPLB-LdEIta (Ld), S.frugiperda cell lines, IPLB-SF21AE (Sf21) and Sf9 (ATCC No. CRL-1711),and Trichloplusia ni cell lines Tn368 and BTI-TN5b1-4 (High Five), allof which are known in the art. Yeast cells can be used for vaccine orpharmaceutical product expression. Production and purification of thegene product may be achieved and enhanced using known techniques.Combining various nucleic acid sequences in a cloning vector can producemosaic peptides.

Examples of appropriate cloning and sequencing techniques, andinstructions sufficient to direct persons of skill through many cloningexercises are found in Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif.; Sambrook et al. (1989) Molecular Cloning—A LaboratoryManual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York; and Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994Supplement). Product information from manufacturers of biologicalreagents and experimental equipment also provide information useful inknown biological methods.

Provided with the T24 peptide sequences described herein, one of skillwill recognize a variety of equivalent nucleic acid molecules thatencode the peptides. This is because the genetic code requires that eachamino acid residue in a peptide is specified by at least one triplet ofnucleotides in a nucleic acid which encodes the peptide. Due to thedegeneracy of the genetic code, many amino acids are equivalently codedby more than one triplet of nucleotides. For instance, the triplets CGU,CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, atevery position where an arginine is to be encoded by a nucleic acidtriplet, the nucleic acid can have any of the triplets that encodearginine. One of skill is thoroughly familiar with the genetic code andits use (for example see chapter 15 of Watson, et al., Molecular Biologyof the Gene (4^(t) Ed., The Benjamin/Cummings Company, Inc., Menlo ParkCalif., 1987), and references cited therein).

Although any nucleic acid triplet or codon that encodes an amino acidcan be used to specify the position of the amino acid in a peptide,certain codons are preferred by certain organisms. In some examples, itis desirable to select codons for elevated expression of an encodedpeptide, for example, when the peptide is purified for use as animmunogenic reagent. Codons can be selected by reference to speciescodon bias tables, which show which codons that are most typically usedby the organism(such as an insect) in which the peptide is to beexpressed. The codons used frequently by an organism are translated bythe more abundant t-RNAs in the cells of the organism. Because thet-RNAs are abundant, translation of the nucleic acid into a peptide bythe cellular translation machinery is facilitated.

Conservative variations of a T24 nucleic acid molecule can yield afunctionally identical construct. For example, due to the degeneracy ofthe genetic code, silent substitutions (substitutions of a nucleic acidsequence that do not result in an alteration in an encoded peptide) arean implied feature of every nucleic acid sequence which encodes an aminoacid. In addition, one of skill will recognize many ways of generatingalterations in a given nucleic acid construct. Such well-known methodsinclude site-directed mutagenesis, PCR amplification using degenerateoligonucleotides, exposure of cells containing the nucleic acid tomutagenic agents or radiation, chemical synthesis of a desiredoligonucleotide (such as in conjunction with ligation or cloning togenerate large nucleic acids) and other well-known techniques. See,Giliman and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328:7314,1987; and Sambrook Ausbel, Berger and Kimmel, all supra.

Modifications to nucleic acids are evaluated by routine screeningtechniques in suitable assays for the desired characteristic. Forinstance, changes in the immunological character of encoded peptides canbe detected by an appropriate immunological assay. Modifications ofother properties such as nucleic acid hybridization to a complementarynucleic acid, redox or thermal stability of encoded proteins,hydrophobicity, susceptibility to proteolysis, or the tendency toaggregate are all assayed according to standard techniques.

Making and Identifying T24 Antigenic Fragments

To identify T24 antigenic fragments, synthetic or recombinant T24peptides are generated. In one example, such peptides are about 200amino acids, about 150 amino acids, about 100 amino acids, about 92amino acids, about 90 amino acids, about 50 amino acids, about 25 aminoacids, about 15 amino acids, or about 10 amino acids. The peptides canbe absorbed to a plastic microwell, nitrocellulose, other membranes, orany other appropriate support. A peptide can be cross-linked to itselfusing a cross-linking agent, such as glutaraldehyde or cross-linked to acarrier protein, such as albumin, keyhole-limpet hemocyanin prior toabsorption to the support. Antibodies present in body fluids frompatients with cysticercosis or monoclonal antibodies specific for T.solium antigens bind the antigenic peptides or polypeptides and aredetected using any immunoassay described below. Reactivity with theantibodies identifies an antigenic fragment.

Smaller T24 peptides can be linked together to form polypeptides rangingin size from 40-200 amino acids using chemical ligation.

T. solium T24 Nucleic Acid Molecules

Nucleic acid molecules encoding a T. solium T24 peptide, and probes orprimers that hybridize to nucleic acid molecules encoding a T24 peptide,are provided. These nucleotides include DNA, cDNA and RNA sequences thatencode for a T24 peptide.

It is understood that all nucleotides encoding a T24 peptide are alsoincluded herein, as long as they encode a polypeptide with therecognized activity, such as the ability to elicit or stimulate animmune response, react with a T24 antibody (such as those found insubjects having a T. solium infection), or combinations thereof. T24nucleotides include sequences that are degenerate as a result of thegenetic code, as long as the T24 amino acid sequence encoded by thenucleotide sequence is not substantially functionally changed.Nucleotide sequences encoding a T24 peptide also includes conservativevariations thereof.

The disclosure provides isolated T24 nucleic acid sequences that containa variation of a T24 sequence, such as a variant T24 nucleic acidsequence. Variants can contain a single insertion, a single deletion, asingle substitution, multiple insertions, multiple deletions, multiplesubstitutions, or any combination thereof (such as a single deletiontogether with multiple insertions) as long as the peptide encodedthereby retains T24 activity. It will be understood by those ordinarilyskilled in the art that the T. solium T24 polypeptides described hereinare also encoded by sequences substantially similar to the nucleic acidsequences provided herein.

In a particular example, a n24 nucleic acid includes SEQ ID NO: 1,nucleotides 33-707 of SEQ ID NO: 1, or nucleotides 342617 of SEQ ID NO:1, or sequences including at least 85%, at least 90%, at least 95%, orat least 98% sequence identity to SEQ ID NO: 1, nucleotides 33-707 ofSEQ ID NO: 1, or nucleotides 342-617 of SEQ ID NO: 1. For example, thefollowing variations can be made to a T24 nucleic acid sequence shown inSEQ ID NO: 1, the “t” at position 53 can be substituted with a “a,” “g,”or “c”; the “c” at position 101 can be substituted with an “a” or “t”;the “c” at position 293 and 341 can be substituted with a “g” “t” or“a;” and the “a” at position 533; can be substituted with a “g.”

In another example, a T24 nucleic acid molecule includes at least 23contiguous nucleotides of SEQ ID NO: 1, for example at least 25, atleast 50, at least 100, at least 200, at least 275, at least 500, atleast 700, or even at least 800 contiguous nucleotides of SEQ ID NO: 1.In some examples, such fragments can selectively hybridize underphysiological conditions to a nucleic acid sequence that encodes a.T24peptide, such as a polynucleotide that encodes SEQ ID NO: 2. The term“selectively hybridize” refers to hybridization under moderately orhighly stringent conditions which excludes non-related nucleotidesequences.

T24 nucleic acid molecules are useful for producing T24 recombinantpolypeptides. Because recombinant polypeptide production can producelarge quantities of polypeptide that require less purification,recombinant polypeptides are often less expensively produced thanpolypeptides produced using traditional isolation or purificationtechniques. A nucleic acid sequence encoding for a T. solium T24 peptidecan be inserted into a vector, into an autonomously replicating plasmidor a virus, into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences such as a plasmid, and recombinantly expressed in a host cell.The term host cell also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof expressing DNA sequences in host cells are well known in the art.

T24 polynucleotide sequences can be inserted into an expression vectorincluding, but not limited to a plasmid, virus or other vehicle that hasbeen manipulated by insertion or incorporation of a T24 nucleic acidsequence. Polynucleotide sequences that encode for T24 can beoperatively linked to expression control sequences. “Operatively linked”refers to a juxtaposition wherein the components so described are in arelationship permitting them to function in their intended manner. Anexpression control sequence operatively linked to a coding sequence isligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. As usedherein, the term “expression control sequences” refers to nucleic acidsequences that regulate the expression of a nucleic acid sequence towhich it is operatively linked. Expression control sequences areoperatively linked to a nucleic acid sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleic acid sequence. Thus expressioncontrol sequences can include appropriate promoters, enhancers,transcription terminators, a start codon in front of a protein-encodinggene, splicing signal for introns, maintenance of the correct readingframe of that gene to permit proper translation of mRNA, and stopcodons. The term “control sequences” includes, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

Exemplary promoters include a minimal sequence sufficient to directtrancription. Also included are promoter elements which are sufficientto render promoter-dependent gene expression controllable for cell-typespecific, tissue-specific, or inducible by external signals or agents;such elements may be located in the 5′ or 3′ regions of the gene.Constitutive and inducible promoters can be used (Bitter et al., Meth.Enzymol. 153:51644, 1987). For example, when cloning in bacterialsystems, inducible promoters such as pL of bacteriophage γ, plac, ptrp,ptac (ptrp-lac hybrid promoter) and the like can be used. When cloningin mammalian cell systems, promoters derived from the genome ofmammalian cells (such as a metallothionein promoter) or from mammalianviruses (such as the retrovirus long terminal repeat; the adenoviruslate promoter; the vaccinia virus 7.5K promoter) can be used. Whencloning in insect cell systems, promoters derived from the genome ofinsect cells (such as OpIE2 for constitutive expression or themetallothionein promoter for inducible expression) can be used.Promoters produced by recombinant DNA or synthetic techniques can alsobe used to provide for transcription of the nucleic acid sequences ofthe disclosure.

The polynucleotide encoding a T24 peptide can be inserted into anexpression vector which contains a promoter sequence which facilitatesthe efficient transcription of the inserted genetic sequence of thehost. The expression vector typically contains an origin of replication,a promoter, as well as specific genes which allow phenotypic selectionof the transformed cells. Vectors suitable for use include, but are notlimited to the T7-based expression vector for expression in bacteria(Rosenberg et al., 1987, Gene 56:125), the pMSXND expression vector forexpression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521,1988), pIZ/V5-His (Invitrogen) and pMIB/V5-His A Invitrogen) forexpression in insect cells, and baculovirus-derived vectors forexpression in insect cells.

Transformation of a host cell with recombinant DNA can be performedusing conventional techniques well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, for example an insect cell, methods oftransfection of nucleic acid molecules include calcium phosphateco-precipitates, conventional mechanical procedures such asmicroinjection, electroporation, insertion of a plasmid encased inliposomes, or use of a viral vectors. Eukaryotic cells can also becotransformed with DNA sequences encoding a T24 peptide, and a secondexogenous DNA molecule encoding a selectable phenotype, such as theherpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein (Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

The resulting recombinantly expressed T24 peptide can be isolated andpurified using conventional methods, for example preparativechromatography and immunological separations involving monoclonal orpolyclonal antibodies.

T24 nucleic acid molecules (and fragments or variants thereof) are alsouseful as nucleic acid probes or primers for the detection of T. soliuminfection in a biological sample, with high sensitivity or specificity.The probes or primers can be used to amplify or detect T. solium larvaenucleic acid molecules in the sample, quantify the amount of T. soliumin the sample, diagnose infection or determine contamination with T.solium , or monitor the progress of therapies used to treat theinfection. T24 nucleic acid molecules are also useful to study the T.solium organism and diseases associated with this organism (such ascystercercosis and neurocystercercosis) and to develop therapies andtreatments for such diseases. In some examples, T24 nucleic acidmolecules are labeled with a detectable label.

Also provided herein are sequences, probes and primers that selectivelyhybridize to the encoding nucleic acid or the complementary, or opposite(or antisense), strand of nucleic acid as those specifically providedherein. Specific hybridization with a nucleic acid can occur with minormodifications or substitutions in the nucleic acid, so long asfunctional, species-specific hybridization capability is maintained.

Hybridization can be achieved under various temperatures and conditions,according to the temperature of dissociation (T_(d)) of the moleculesbeing hybridized and the stringency required for specific binding. Themolecules can be hybridized to one another in any order or at the sameor essentially the same time. Reaction conditions for hybridization ofan oligonucleotide, or polynucleotide, to a nucleic acid sequence varyfrom oligonucleotide to oligonucleotide, depending on factors such asoligonucleotide length, the number of G and C nucleotides, and thecomposition of the buffer utilized in the hybridization reaction.

Moderately stringent hybridization conditions are when the hybridizationis performed at about 42° C. in a hybridization solution containing 25mM KPO₄ (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mnL probe (such as about 5×10⁷ cpm/μg), while the washes areperformed at about 50° C. with a wash solution containing 2×SSC and 0.1%sodium dodecyl sulfate (SDS).

Highly stringent hybridization conditions are when the hybridization isperformed at about 42° C. in a hybridization solution containing 25 mMKPO4 (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured,sonicated salmon sperm DNA, 50% formamide, 10% dextran sulfate, and 1-15ng/mL probe (such as about 5×10⁷ cpm/μg), while the washes are performedat about 65° C. with a wash solution containing 0.2×SSC and 0.1% SDS.

If used as primers, T24 nucleic acid molecule compositions can includeat least two nucleic acid molecules that hybridize to different regionsof the T24 target molecule so as to amplify a desired region of T24.Depending on the length of the probe or primer, the target region canrange between 85% complementary bases and full complementarity and stillhybridize under stringent conditions. In specific examples, thehybridizing nucleic acid probes or primers described herein have atleast 85%, at least 90%, at least 95%, at least 97%, at least 98%, or atleast 99% complementarity with the segment of the sequence to which theyhybridize, for instance 85% or more. For the purpose of determining thepresence of T. solium , the degree of complementarity between thehybridizing nucleic acid (probe or primer) and the sequence to which ithybridizes is at least enough to distinguish hybridization with anucleic acid from other organisms. In particular examples, a probe orprimer is a DNA molecule having a length of 20 to 40 nucleotides, suchas 25 to 35 nucleotides.

Amplification of synthesized T24 DNA can be detected by any method usedin the art. Exemplary detection methods include Southern blothybridization, visualization of DNA amplification products of specificmolecular weight on ethidium bromide stained agarose gels, measurementof the incorporation of radiolabeled nucleotides into the synthesizedDNA strand by autoradiography or scintillation measurement, and ELISAmodified for the capture of a detectable moiety bound to the amplifiedDNA. One particular detection method is hybridization of the amplifiedDNA to an internal specific oligo-probe using ELISA, Southern blothybridization or similar methods.

Labeled T24 T. solium Polypeptides

When labeled with a detectable biomolecule or chemical, a T. solium T24peptide, including antigenic fragments thereof, are useful fordiagnostic methods and assays described below. Various types of labelsand methods of conjugating the labels to the polypeptides are wellknown. Several exemplary labels are set forth below.

For example, a T24 peptide can be conjugated to a radiolabel such as,but not limited to, ³²P, ³H, ¹⁴C, ³⁵S, ¹²⁵I, or ¹³¹I. Detection of alabel can be by methods such as scintillation counting, gamma rayspectrometry or autoradiography.

Bioluminescent labels, such as derivatives of firefly luciferin, canalso be used. The bioluminescent substance is covalently bound to thepolypeptide by conventional methods, and the labeled polypeptide isdetected when an enzyme, such as luciferase, catalyzes a reaction withATP causing the bioluminescent molecule to emit photons of light.

Fluorophores can also be used as labels. Examples of fluorophoresinclude fluorescein and derivatives, phycoerythrin, allo-phycocyanin,phycocyanin, rhodamine, and Texas Red. Fluorophores can be excited usingan appropriate laser or filter and detected using a fluorescencedetector.

The T24 polypeptides can also be labeled with a chromogen to provide anenzyme or affinity label. For example, the polypeptide can bebiotinylated so that it can be utilized in a biotin-avidin reaction,which can also be coupled to a label such as an enzyme or fluorogen.Alternatively, the polypeptide can be labeled with peroxidase, alkalinephosphatase or other enzymes giving a chromogenic or fluorogenicreaction upon addition of substrate. Additives such as5-amino-2,3-hydro-1,4-phthalazinedione (also known as Luminol™) (Sigma,St. Louis, Mo.) and rate enhancers such as p-hydroxybiphenyl (also knownas p-phenylphenol) (Sigma) can be used to amplify enzymes such ashorseradish peroxidase through a luminescent reaction; and luminogenicor fluorogenic dioxetane derivatives of enzyme substrates can also beused. Such labels can be detected using enzyme-linked immunoassays(ELISA) or by detecting a color change with the aid of aspectrophotometer. In addition, peptides can be labeled with colloidalgold for use in immunoelectron microscopy in accordance with methodswell known to those skilled in the art.

The diagnosis of an infection by T. solium larvae can be determined bylabeling a T24 peptide as described above, incubating the labeledpeptide with a sample, and detecting the label. The presence ofdetectable label indicates that the sample contains (or has been exposedto) T. solium larvae, while the absence of detectable label indicatesthat the sample does not contain (or has not been exposed to) T. soliumlarvae.

Detection of T24 T. solium Antibodies

Many methods are known for detecting and quantifying a component, suchas an antibody in a mixture, or for measuring its amount. Immunoassays,which employ peptides that bind specifically to antibodies of interest,can be used. These methods permit detection (or quantification) of T.solium antibodies present in a subject to indicate the presence or levelof T. solium infection, and in some examples the diagnosis of a diseaseor condition associated with such infection, for example cysticercosis.Exemplary methods involve contacting a biological sample containing theantibody with a known excess amount of peptide specific for theantibody, such as a disclosed T24 peptide, separating bound from freeantibody, and determining the amount of one or the other, for example bydetecting a label on the T24 peptide or by incubating with a secondaryantibody. The second antibody can be labeled to aid in the determinationof the amount of bound analyte as described above.

In one example, the diagnostic method uses a rapid immunochromatographicdiagnostic test (card test) assay. In a further example, the diagnosticmethod is a rapid card test assay including one or more larval T. soliumprotein antigens, such as T24, or an antigenic fragment thereof.

The assay methods can include the use of synthetic or recombinant T24 T.solium peptides as described above, as well as fusions, fragments orvariants of the T. solium T24 peptide described herein as long as thefusions, fragments or variants retain immunogenic activity, the abilityto immunoreact with a T24 antibody, or combinations thereof. Thesefusions, fragments or variants include T24 peptides with antigenicactivity that have amino acid substitutions or have other moleculesattached to amino acid functional groups as described above. In someexamples, the assay methods include the use of one or more othersynthetic or recombinant T. solium larval peptides, or fusions,fragments or variants thereof.

These other polypeptides include, but are not limited to, gp50, gp39-42,gp21, gp18, gp14 and gp13 (see U.S. Pat. No. 5,354,660, hereinincorporated by reference).

An immunoassay for the detection of T. solium in a sample can beperformed as follows. To detect T. solium antibodies in a sample, suchas T24 antibodies, the sample is incubated with one or more T. soliumrecombinant or synthetic peptides, such as the T24 peptides disclosedherein. The peptide can be labeled or conjugated to a solid phase beador particle as described herein. The labeled peptide is detected usingwell known techniques for detection of biologic molecules, such asimmunochemical or histological methods. For example, immunologicaltechniques employing monoclonal or polyclonal antibodies to thepolypeptide, such as enzyme linked immunosorbant assays,radioimmunoassay, chemiluminescent assays, or other types of assaysinvolving antibodies can be used.

The sample potentially containing the T. solium antibodies to bedetected can be collected or obtained from a subject using methods wellknown to those skilled in the art. The sample can be obtained from anybiological source, such as blood serum, blood plasma, urine, spinalfluid, saliva, fermentation fluid, lymph fluid, tissue culture fluid andascites fluid. In addition, the sample can be diluted, purified,concentrated, filtered, dissolved, suspended, or otherwise manipulatedprior to immunoassay.

In general, binding assays rely on the binding of analyte by analytereceptors to determine the concentrations of analyte in a sample. Theseimmunoassays can be described as either competitive or noncompetitive.Non-competitive assays generally utilize analyte receptors insubstantial excess over the concentration of analyte to be determined inthe assay. Sandwich assays are examples of non-competitive assays, whichinclude one analyte receptor frequently bound to a solid phase and asecond analyte receptor labeled to permit detection. The analyte firstbinds to the analyte receptor bound to a solid phase and the secondlabeled analyte receptor is then added to facilitate quantitation of theanalyte. Bound analyte can easily be separated from unbound reagents,such as unbound labeled first analyte receptors, due to the use of ananalyte receptor bound to a solid phase.

Competitive assays generally involve a sample suspected of containinganalyte, an analyte-analogue conjugate, and the competition of thesespecies for a limited number of binding sites provided by the analytereceptor. Competitive assays can be homogeneous or heterogeneous. Inhomogeneous assays all reactants participating in the competition aremixed together and the quantity of analyte is determined by its effecton the extent of binding between analyte receptor and analyte-conjugateor analyte analogue-conjugate. The signal observed is modulated by theextent of this binding and can be related to the amount of analyte inthe sample.

Methods are also disclosed for the diagnosis or prognosis ofcysticercosis. A determination of the presence of larval T. soliumantibodies, such as a T24 antibody, can be made using the recombinant orsynthetic T24 polypeptides or antigenic fragments thereof describedherein as reagents in assays using assay techniques that are well knownto those skilled in the art and include methods such as rapidimmunochromatographic diagnostic tests, Western blot analysis,radioimmunoassay and ELISA assays. For example, a sample can beincubated with a T. solium protein, such as a T24 protein, for a timesufficient to allow the protein and antibodies in the sample to bind,and detecting binding of the protein with the antibodies. The proteincan be labeled to permit detection of protein:antibody complexes.Alternatively, protein:antibody complexes can be detected by using alabeled secondary antibody. The presence of detectable protein:antibodycomplexes indicates that the subject has cysticercosis, while theabsence of detectable protein:antibody complexes indicates that thesubject does not have cysticercosis. A decrease in detectableprotein:antibody complexes can indicate that the subject is recoveringfrom cysticercosis. Samples can be obtained, for instance, from theblood, cerebrospinal fluid, urine, saliva, or tissues of a mammal, suchas a human or pig.

Detection of T24 T. solium Proteins and Nucleic Acid Molecules

The disclosure herein of T24 protein and nucleic acid sequences permitsone to detect such sequences in a sample. These methods permit detection(or quantification) of T. solium protein and nucleic acid sequencespresent in a subject to indicate the presence or level of T. soliuminfection, and in some examples the diagnosis of a disease or conditionassociated with such infection, for example cysticercosis.

Exemplary methods include contacting a biological sample containing T24nucleic acid sequences with a probe or primer that can recognize the T24nucleic acid sequence. In some examples, nucleic acid molecules presentin the sample are first isolated prior to contacting with the probe orprimer. For example, nucleic acid molecules can be isolated andtransferred to a membrane (such as nitrocellulose), which is incubatedwith a probe that recognizes T24 sequences. In one example, a probecontaining a detectable label is incubated with the sample for a periodtime to allow hybridization between the probe and T24 nucleic acidmolecules. The presence of probe:T24 nucleic acid molecule complexes isthen determined by detecting the label on the probe (wherein thepresence of such complexes indicates that the subject has a T. soliuminfection, cysticercosis, neurocysticercosis, or combinations thereof).In a particular example, primers are used to amplify a T24 nucleic acidsequence from a sample. For example, PCR or RT-PCR can be used, and thepresence of resulting amplicons determined. The presence of ampliconsindicates that the subject has a T. solium infection, cysticercosis,neurocysticercosis, or combinations thereof.

Other methods involve contacting a biological sample containing T24protein sequences with an antibody that can recognize the T24 protein.For example, the sample can be incubated with an antibody thatrecognizes a T24 protein, and the presence of antibody:protein complexesdetermined, for example by using microscopy, a spectrophotometer or flowcytometry. The presence of antibody:protein complexes indicates that thesubject has a T. solium infection, cysticercosis, neurocysticercosis, orcombinations thereof. In some examples, the antibody includes a labelthat permits detection of the complexes. In other examples, a labeledsecondary antibody is used to detect the complexes.

In some examples, proteins present in the sample are first purifiedprior to contacting with an antibody. For example, the sample can besubjected to SDS-PAGE, and the proteins transferred to a membrane, suchas nitrocellulose. The membrane is then incubated with the antibody, andthe presence of antibody:protein complexes determined. The presence ofantibody:protein complexes indicates that the subject has a T. soliuminfection, cysticercosis, neurocysticercosis, or combinations thereof.In some examples, the antibody includes a label that permits detectionof the complexes. In other examples, a labeled secondary antibody isused to detect the complexes.

Control samples can include positive controls, for example sera known tobe infected with T. solium, or a sample containing known T24 nucleicacid molecules or proteins. Control samples can include negativecontrols, for example sera known to be free of T. solium, a sampleinfected with another pathogen, or a sample that does not containnucleic acid molecules or proteins.

Kits for Detecting T. solium or for Diagnosing a T. solium-AssociatedCondition

Kits for detecting the presence or quantity of T. solium in a sample, orfor diagnosis a T. solium-associated disease or condition, are alsoprovided. The kits can be in any configuration useful for performing oneor more of the assays described herein for the detection of T. solium inbiological samples or for the detection or monitoring of T. soliuminfection in a patient or carrier. The kits are convenient in that theysupply many, if not all, of the essential reagents for conducting anassay for the detection of T. solium in a biological sample. Thereagents can be pre-measured and contained in a stable form in vesselsor on a solid phase in or on which the assay can be performed, therebyminimizing the number of manipulations carried out by the individualconducting the assay. In addition, the assay can be performedsimultaneously with a standard included with the kit, such as apredetermined amount of larval T. solium antigen or antibody, so thatthe results of the test can be validated or measured.

In certain examples, the kits contain recombinant or synthetic T24 T.solium peptides for the detection of T24 T. solium antibodies, orinclude T24 nucleic acid molecules to detect or amplify T. soliumnucleic acid molecules present in a sample. The kit can further includeone or more additional recombinant or synthetic T. solium larvalpolypeptides or nucleic acid molecules described herein. The kits canadditionally contain the appropriate reagents for binding thepolypeptides to the antibodies or hybridizing the nucleic acid moleculesto their respective T. solium complementary nucleic acid molecules inthe sample as described herein and reagents that aid in detecting theantibody-polypeptide or nucleic acid molecule complexes. The kits cancontain equipment for safely obtaining the sample, a vessel forcontaining the reagents, a timing means, a buffer for diluting thesample, and a colorimeter, reflectometer, or standard against which acolor change may be measured.

In specific examples, the reagents, including the T24 peptides, arelyophilized, for instance in a single vessel. Addition of aqueous sampleto the vessel results in solubilization of the lyophilized reagents,causing them to react. In certain specific examples, the reagents aresequentially lyophilized in a single container, in accordance withmethods that minimize reaction by the reagents prior to addition of thesample. Such methods are well known to those of ordinary skill in theart.

In some examples, the T24 peptides are present on a solid substrate,such as a membrane (for example nitrocellulose) or polystyrene. Suchsubstrates can be incubated with a sample (such as a sample suspected ofcontaining T24 antibodies), and the presence of a peptide:antibodycomplex determined.

Specific examples of assay kits include, but are not limited to,reagents to be employed in one or more of the following methods:competitive and noncompetitive assays, radioimmunoassay, bioluminescenceand chemiluminescence assays, fluorometric assays, sandwich assays,immunoradiometric assays, dot blots, enzyme linked assays includingimmunoblots and ELISAs, and immunocytochemistry. Materials used inconjunction with these techniques include, but are not limited to,microtiter plates, antibody (or T24 protein) coated strips or dipsticksfor rapid monitoring of urine or blood. For each kit, the range,sensitivity, precision, reliability, specificity, and reproducibility ofthe assay are established.

In another example, the assay kit uses immunoblot techniques andprovides instructions and one or more synthetic or recombinant larval T.solium polypeptides, such as T24, conjugated to a detectable molecule.The kit can be used to detect and measure T. solium in biological fluidsand tissue extracts of animals and humans to diagnose or monitorcysticercosis or neurocysticercosis.

T24 Sequences as Immunogenic Compositions

Methods are provided for treating a subject having cysticercosis orneurocysticercosis, or preventing the development of cysticercosis orneurocysticercosis following T. solium infection. The method includesadministering to the subject a therapeutically effective amount of oneor more T24 epitopes (or nucleic acid encoding such an epitope), therebytreating cysticercosis or neurocysticercosis, for example by decreasingone or more symptoms associated with cysticercosis orneurocysticercosis. The T24 eptiope(s) can be administered in thepresence of pharmaceutically acceptable carrier, alone or in thepresence of an agent that aids in stimulation of the immune response,such as an adjuvant. In one example, the susceptibility of the subjectto T. solium infection, such a& a human or pig, is determined prior toadministering a therapeutically effective amount of a T24 epitope.

Also disclosed are methods for eliciting an immune response in asubject. The method includes administering to the subject atherapeutically effective amount of one or more T24 epitopes (or nucleicacid encoding such an epitope), thereby eliciting an immune response ina subject. Specific, non-limiting examples of an immune response are a Bcell or a T cell response. Methods of generating antibodies specific fora T24 antigen, are disclosed. The method includes administering to asubject one or more T24 antigens, such as SEQ ID NO: 2 or amino acids104-195 of SEQ ID NO: 2. In one example, the subject is an experimentalanimal, such as a mouse or rabbit. In another example, the subject is amammal, such as a pig or human.

In forming a pharmaceutical composition for eliciting an immune responsein a subject, or for treating cysticercosis or neurocysticercosis, oneor more T24 epitopes (at a therapeutically effective amount), alone orin combination with other agents, is utilized. T24 epitope variants,fragments, and fusions can be employed in the pharmaceuticalcompositions, and can include one or more amino acid additions, aminoacid deletions, amino acid replacements, or by isostereomer (a modifiedamino acid that bears close structural and spatial similarity to theoriginal amino acid) substitutions, and isostereomer additions, so longas the sequences are recognized by, or can generate, and immune cell.For example, a variant of SEQ ID NO: 2, such as amino acids 104-195 ofSEQ ID NO: 2, will be recognized by an immune cell that recognizes SEQID NO: 2. In a particular example, such variants, fragments, andfusions, provide an advantage, such as increasing the solubility orimmungenicity of the eptitope, or easing linking or coupling of theepitope. In one example, the peptides included in the pharmaceuticalcomposition can form neutralizing antibodies to a T24 epitope.

The disclosed T24 peptides can be engineered to include other aminoacids (to generate a fusion protein), such as residues of variousmoieties, such as additional amino acid segments or polysaccharides.Examples include, but are not limited to, moieties which augment orinduce antigen processing, epitope stability or manufacture, or deliverywithin the body to sites appropriate for immunization or recognition byimmune cells. In addition, an amino acid chain corresponding to anadditional antigen or immunogen can be included. Thus, an immuneresponse to more than one antigen can be induced by immunization.Specific non-limiting examples of antigens or immunogens include, butare not limited to, other T. solium larval proteins that increase orprovoke CD4⁺ T-cell (helper T-cell) responses supportive of a T24 immuneresponse. These additional amino acid sequences can be of varyinglength.

In some examples, it is desirable to combine two or more T24 epitopesthat contribute to stimulating specific immune responses in one or moresubjects or histocompatibility types. The epitopes in the compositioncan be identical or different, and together they may provide equivalentor greater biological activity than the parent epitopes(s). For example,multiple epitopic peptides can be combined in a “cocktail” to provideenhanced immunogenicity, and peptides can be combined with peptideshaving different MHC specificities. Such compositions can be used toeffectively broaden the immunological coverage provided by therapeutic,immune stimulatory composition or diagnostic methods and compositions.

In some examples, epitopic peptides are linked with or without a spacermolecule to form polymers (multimers), or can be formulated in acomposition without linkage, as an admixture. Where the same peptide islinked to itself, thereby forming a homopolymer, a plurality ofrepeating epitopic units are presented. When the peptides differ,heteropolymers with repeating units are provided.

Linkages for homo- or hetero-polymers or for coupling to carriers andadjuvants can be provided in a variety of ways, such as through covalentlinkages between epitopic peptides or noncovalent linkages capable offorming intermolecular and intrastructural bonds. When present, thespacer can include relatively small, neutral molecules, such as aminoacids or amino acid mimetics, which are substantially uncharged underphysiological conditions and may have linear or branched side chains.Particular examples of spacers include on or more alanine or glycineresidues, or other nonpolar amino acids or neutral polar amino acids.Spacers can be either homo- or hetero-oligomers and can include one ortwo residues, more typically three to six residues. Spacers can beattached to epitopic peptides at the C-terminus, N-terminus or a sidechain of one or more of the amino acids. Examples of crosslinking agentswhich can be used to interconnect a plurality of epitopes includecrosslinking agents which have as their functional group an aldehyde(such as glutaraldehyde), carboxyl, amine, amido, imido or azidophenylgroup. In particular, butyraldehyde can be used as a crosslinking agent,a divalent imido ester or a carbodiimide.

In another example, cysteine residues can be added at the amino- andcarboxy-termini to permit formation of bonds between peptides viacontrolled oxidation of the cysteine residues. Heterobifunctionalagents, which generate a disulfide link at one functional group end anda peptide link at the other, includingN-succidimidyl-3-(2-pyridyldithio) proprionate (SPDP) may also beemployed. A variety of such disulfide/amide, forming agents are known(For example, Immun. Rev. 62:185, 1982). Other bifunctional couplingagents form a thioether rather than a disulfide linkage. Many of thesethioether forming agents are commercially available and include reactiveesters of 6-maleimidocaproic acid, 2 bromoacetic acid, 2-iodoaceticacid, and 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid. In thesereagents, the carboxyl groups can be activated by combining them withsuccinimide or 1-hydroxy-2-nitro-4-sulfonic acid, sodium salt. Onecoupling agent is succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Ideally, the linkage does notsubstantially interfere with the immunogenicity of the linked epitopicpeptides.

A particular example of a fusion protein, which includes one or more T24epitopic peptide sequences, can be used to present the epitopic peptidesto a subject. For example, a recombinant HBV surface antigen protein isprepared in which the HBenv amino acid sequence is altered so as to moreeffectively present epitopes of peptide regions described herein tostimulate an immune response. By this means a polypeptide mayincorporate several epitopes. Coding sequences for peptides of thelength contemplated herein can be synthesized by chemical techniques,for example, the phosphotriester method of Matteucci et al. (J. Am.Chem. Soc. 103:3185, 1981). The coding sequence can then be providedwith appropriate linkers and ligated into expression vectors commonlyavailable in the art, and the vectors used to transform suitable hoststo produce the desired fusion protein as disclosed herein and known inthe art.

In one example, the disclosed T24 epitopes, such as SEQ ID NO: 2 or afragment thereof, such as amino acids 104-195 of SEQ ID NO: 2, isobtained from natural sources, chemically synthesized, or producedrecombinantly. The T24 protein is subjected to selective proteolysis,for example by splitting the protein with chemical reagents or enzymes.The length of the amino acid sequence produced can depend on the methodof producing the sequence. If the sequence is made by assembling aminoacids by chemical means, the sequence ideally does not exceed, forexample, about 50, about 40, or about 30 amino acids. If the syntheticpeptide is made by translating a nucleic acid, the peptide can be anylength, including, for example, about 100 amino acids or more. However,the peptide can also be shorter, for example, no more than 50, no morethan 40, no more than 20, no more than 10, no more than 9, or no morethan 8 amino acids, for example about 8-50 amino acids.

This disclosure is further illustrated by the following Examples, whichare not to be construed in any way as imposing limitations upon thescope of the disclosure. It is understood that resort may be had tovarious other embodiments, modifications, and equivalents thereof that,after reading the description herein, may suggest themselves to those ofordinary skill in the art, without departing from the spirit of thepresent disclosure or the scope of the appended claims.

EXAMPLE 1 Cloning and Analysis of T24 kDa from T. solium

This example describes the methods used to purify and clone T24 (LLGP24)from Triton X-114 extracted T. solium cysticerci or from lentil lectinpurified glycoprotein (LLGP). Although the molecular weight of severalT. solium larval proteins was previously known, the sequence of T24 hadnot been identified. Purification and cloning of T24 proved difficult,because several proteins (about nine) co-migrated at the same positionon SDS-PAG. Therefore, the T24 protein was not previously substantiallypurified, even though its molecular weight on an SDS-PAG was known. Itis likely that previous preparations of T24, even on SDS-PAG, were onlyabout 30% pure by weight, due to the contaminating proteins thatco-migrate in this region. Determining the sequence of T24 is importantto permit recombinant expression of T24 to allow for the development ofa less-expensive diagnostic assay for T. solium infection.

Purification and Cloning from Triton X-114 extracted T. soliumcysticerci

Approximately 200 mg of T. solium cysts were homogenized, extracted withthe detergent Triton X-114, and the proteins were separated by phasepartitioning into an aqueous phase and a detergent phase. Proteins inthe detergent phase are membrane proteins. The proteins in each phasewere analyzed by silver staining and western blotting (FIGS. 1 and 2A).Proteins immunoreactive with TSC-1 (pool sera from threeparasitologically confirmed cysticercosis patients) were observed in thedetergent phase at 24-, 42-, and 50-kDa. Their size, immunoreactivitywith TSC-1 and a panel of cysticercosis positive sera, andsusceptibility to DTT reduction were confirmed by silver staining andwestern blotting.

Approximately 200 μg of the fraction containing T24 and T42 were acetoneprecipitated, resolved by SDS-PAGE, blotted onto PVDF membrane, and thebands at 24- and 42-kDa excised using a corresponding western blot asthe guide. Approximately 390 μg of the same fraction was acetoneprecipitated, resolved by SDS-PAGE, stained with Colloidal CoomassieStain, and the bands at 24- and 42-kDa excised and sequenced.

The N-terminus of the proteins on PVDF was sequenced by Edmandegradation. The N-terminus sequences of both proteins were identical.The proteins in the gel slice were digested with trypsin and the trypticpeptides were analyzed by MS/MS spectra using the Sequest algorithm.However, the MS[MS data were not informative. The four tryptic peptidesof T24 from the gel slice were subsequently sequenced using Edmandegradation. Five internal peptide sequences were obtained by denovoMS/MS sequencing of the T42 sample. All five of the T42 peptidesequences are within the coding sequence of T24.

A search of the BLAST nr and est databases with the N-terminal sequenceand with one of the internal sequences from T24 identified severalEchinococcus granulosus EST sequences (Genbank Accession Nos: BI244014,BF643023, BQ17343 1, BQ173376, BG173191, and BQ173064). The sequencesshown in Genbank Accession Nos: BI244014 and BF643023 appear to overlapand form an ORF beginning with the initiating methionine. All of thepeptide sequences share homology to the E. granulosus EST sequences.

Purification and Cloning from LLGP

T24/T42 (LLGP24/LLGP42) was purified from LLGP as follows. Approximately2.5 mg of LLGP, lot Ts10322, were subjected to preparative SDS-PAGEusing a Model 491 Prep Cell (BioRad). One hundred fifty fractions werecollected and analyzed by silver staining and western blotting. Based onsize, fractions 40-48 were pooled to form the LLGP24 pool and fractions78-90 were pooled to form the LLGP42 pool. Proteins were concentrated byacetone precipitation. Their size, immunoreactivity with a pool ofcysticercosis infection sera (TSC-1), and susceptibility to DTTreduction were confirmed by silver staining and western blotting.

Approximately half of each pool was resolved by SDS-PAGE, blotted ontoPVDF membrane, and the bands at 24- and 42-kDa were excised using acorresponding western blot as the guide. The other half of each pool wasresolved by SDS-PAGE, stained with Colloidal Coomassie Stain(Invitrogen), and the bands at 24- and 42-kDa were excised. TheN-terminus of the proteins on PVDF was sequenced by Edman degradation.The proteins in the gel slice were digested with trypsin and the trypticpeptides were analyzed by MS/MS spectra using the Sequest algorithm.This was followed by Edman degradation sequencing of four trypticpeptides for LLGP24 and one tryptic peptide for LLGP42.

Results

Sequences from several non-T24 proteins were identified frompurification from LLGP, when N-terminal sequencing, Edman degradation,or MS/MS spectra were used. This made identification of T24 sequencesdifficult. The MS/MS spectra data provided several sequences, including:T24 and the 8-kDa family of proteins (Ts14, Ts18var1, Ts low molecularweight antigen 1, Ts low molecular-weight antigen 2), but did notgenerate any new sequences. T24 would not have been identified in thesequences of the LLGP proteins at 24 kDa had it not already been clonedfrom the triton-extracted T24, because MS/MS spectra and the Sequestalgorithm can only identify spectra from known sequences. Triton X-114purified T24 and T42 were sequenced. Edman degradation sequencing offour tryptic peptide fragments from T24 and of the N-terminus wasperformed. De novo MS sequencing of T42 gave five sequences and all werewithin the cloned T24 cDNA. In the final cloned T24, nine of these 10peptides sequences, five from T24 sequences and five from T42sequencing, are within the predicted mature protein sequences. The tenthcould not be identified.

The results for LLGP are summarized in Tables 1 and 2. TABLE 1 Sequencesidentified from LLGP-24 band N-terminal MS/MS spectra of Edmandegradation of sequencing Tryptic Peptides Tryptic Peptides Ts14 (matureT24, 16 spectra, 9.3e4 peptide 67 - 8-kDa N terminus) Ts14, 13 spectra,3.3e4 protein (matches with AJ012670 Ts18var1, 7 spectra, Ts14 and TsRS2(NC-9) 4.2e4 sequences) (internal AB044080 (TsRS2 peptide 71 - 8-kDafragment) clade), 4 spectra, 1.2e4 protein (matches with AJ012670(NC-9), 7 only Ts18 sequences) spectra, 4.7e4 peptide 86 - AJ012670AB044082 (TsRS1 (NC-9) and a clade), 5 spectra, 5.6e4 secondary sequenceAJ012669 (NC-3), 5 with no hits spectra, 1.0e4 peptide 94 - AJ012670Echino P-29, 4 spectra, (NC-9), a secondary 4.8e3 sequence that matchesAF523312 (oncosphere- lectin, and a tertiary specific antigen, relatedsequence with no hits to 8-kDa proteins), 2 spectra, 2.7e3

TABLE 2 Sequences identified from LLGP-42 band N-terminal MS/MS spectraof Edman degradation of sequence* Tryptic Peptides Tryptic Peptides Ts14(mature Ts18var1, 6 spectra, AJ012670 (NC-9) N terminus) 1.1e4 AJ012670Echino P-29, 5 spectra, (NC-9) 2.5e3 (internal Ts14, 3 spectra, 8.7e2fragment) T24, 1 spectra, 4.7e2 AJ012670 (NC-9), 1 spectra, 2.0e3

Based on the N-terminal sequence and MS/MS spectra results, it appearsthat the 8-kDa proteins are components of LLGP-24 and LLGP-42. Thepredominant 8-kDa proteins were Ts14 and Ts18var1; however, GenbankAccession Nos: AB044082 (TsRS1 c1ade) and AB044080 (TsRS2 c1ade) werealso observed. T24 was the predominant protein in the MS/MS spectra ofLLGP-24, and was also observed in the MS/MS spectra of LLGP42.

AJ012670 (NC-9) was a significant component present in LLGP-24 andLLGP42. However, it is known that the sensitivity of a GST-NC-9recombinant protein for detecting cysticercosis is only 33.3%. E.granulosus P-29 (Genbank Accession NO. AF078931) was to a lesser extenta significant component of LLGP-24 and LLGP42. P-29, NC-9, and NC-3 werealso observed in the MS/MS spectra of T24. P-29 was observed in theMS/MS spectra of T42.

These results also demonstrate that T24 is the impure componentpreviously identified as LLGP-24. LLGP-24 is a well-characterizedcomponent of the LLGP diagnostic antigen used for more than ten years asthe diagnostic gold-standard for the disease cysticercosis, caused bythe larval stage of T. solium. LLGP is purified from urea-solubilized T.solium cysts (harvested from pig muscle). The urea-solubilized proteinsare dialyzed into the appropriate buffer and run on a lentil lectinaffinity column. The T24 diagnostic antigen is the bound fraction fromthis column. However, as described above, T24 can also be obtained fromthe detergent soluble fraction of a Triton X-114 extraction of cysts. Inaddition, the sequencing data indicates that T42 is a dimer of T24.LLGP42 is a component of the diagnostic antigen band 39-42.

Based on the sequences obtained using these methods, T24 was clonedusing the protein sequence data obtained from N-terminal and internalsequences of the Triton-extracted protein. Using a degenerate forwardprimer based on the N-terminal sequence and degenerate reverse primersbased on the internal peptide sequence, a portion of the cDNA wasamplified using a T. solium cDNA library prepared from cysts as thetemplate. The full-length cDNA was obtained by random prime labeling ofthe partial sequence and probing the cDNA library. Plaques identified aspositive by autoradiography were purified. Plaques with the largestinserts were sequenced. Additional 5′ sequence was determine using 5′RACE. The cDNA and protein sequences for a T24 are shown in SEQ ID NOS:1 and 2, respectively.

EXAMPLE 2 Recombinant Expression of T24

This example describes methods used to express T24 (SEQ ID NO: 2)recombinantly in insect cells.

Full-length T24, amino acids 1 through 225 (SEQ ID NO: 2), wereexpressed recombinantly using an insect cell expression system. Anucleic acid sequence encoding SEQ ID NO: 2 (nucleotides 33-710 of SEQID NO: 1) was cloned into pMT/BiP/V5-His A (Invitrogen, Carlsbad,Calif.) using standard molecular biology methods. Briefly, the vector,was cut with Bgl II and Kpn I and treated with phosphatase. The vectorand insert were ligated, generating pMT/BiP/T24, and then transformedinto E. coli TOP10 cells (other cells can be used, such as E. coliDH5αT1 cells). Recombinant cells were selected with carbenicillin. Thecorrect construction of the clone used for expression was confirmed bysequencing.

The vector generated above (pMT/BiP/T24) was purified from TOP10 cells(Invitrogen) using Qiagen midi-prep kit for plasmid DNA purification,and then transiently expressed in the insect cell line D. melanogasterSchneider 2 (S2) (ATCC, Manassas, Va.) using serum-free medium.Transfection was performed using calcium phosphate. After 72 hours, 500μM copper sulfate was added to the culture to induce gene expression.The cultures were sampled at 24-hour intervals from 0 to 96 hourspost-induction. The culture medium was centrifuged and separated intocell pellet and supernatant. Expression of T24 was observed in thesupernatant beginning at 24 hours post-induction increasing at 48 hoursand then holding steady at 72 and 96 hours post-induction.

To determine if recombinant T24 is immunoreactive with T24 antibodies,the following methods were used. The 96-hour supernatant generated abovewas loaded on a western blot at 0.625 μ/mm and strips made. Therecombinant T24 protein migrates at a little less than 30 kDa. T24 wasreactive with sera known to be infected with T. solium. Because thegenus Echinococcus is the most closely related genus to Taenia, serumfrom people infected with Echinococcus is potentially cross-reactivewith Taenia antigens. However, recombinant T24 (as well asTriton-extracted T24 from Example 1) was not reactive with seruminfected with Echinococcus granulosus. Therefore, T. solium T24 isspecies-specific even though its sequence is similar to Echinococcussequences.

EXAMPLE 3 Immunoreactive Fragment of T24

This example describes methods used to generate a 92 amino acid fragmentof T24, and methods used demonstrate that the fragment recognizes T24antibodies and thus has immunoreative activity.

The hydrophilic portion of T24 (amino acids 104 through 195 of SEQ IDNO: 2), referred to herein as T24H, was recombinantly expressed using aninsect cell expression system. This 92 amino acid fragment of T24 wasamplified using Pfu polymerase. The forward primer was designed with aBgl II restriction enzyme site, the reverse primer with a Kpn I site andtwo stop codons. The cloning was designed so that the insert would bein-frame with the BiP secretory signal of the vector. The vector,pMT/BiP/V5-His A Invitrogen), was cut with Bgl II and Kpn I and treatedwith phosphatase. The vector and insert were ligated, generatingpMT/BiP/T24H, and then transformed into E. coli TOP10 cells. Recombinantcells were selected with carbenicillin. The correct construction of theclone used for expression was confirmed by sequencing.

The vector generated above was purified from TOP10 cells as described inExample 2 and then transiently expressed in the insect cell line S2 (seeExample 2) using serum-free medium. Transfection and induction wereperformed as described in Example 2. The cultures were sampled at24-hour intervals from 0 to 96 hours post-induction. The culture mediumwas centrifuged and separated into cell pellet and supernatant.Expression of T24H was observed in the supernatant beginning at 24 hourspost-induction with maximum expression at 96 hours.

To determine if T-24H is immunoreactive with T24 antibodies, thefollowing methods were used. The 96-hour supernatant generated above wasloaded on a western blot at 0.625 μg/mm and strips made. The recombinantT24H protein migrates at about 10 kDa. T24H was reactive with sera knownto be infected with T. solium, and with goat anti-native T24 antibodies.Out of 21 infection sera that react with native T24, 20 also reactedwith recombinant T24H, indicating a sensitivity of 95%. In addition, outof 13 T. solium negative sera, 10 from other parasitic infections (EMEUS377) and 3 NHS, 12 were negative indicating a specificity for T24H of92%. These results indicate that T24H can distinguish Taenia fromEchinococcus.

In addition, both recombinant full-length T24 (Example 2) andrecombinant T24H were sensitive to reduction with DTT, similar to thenative protein. After treatment of the recombinant proteins with DTT,all antibody reactivity with infection sera was lost. 30 In summary,these results demonstrate that a 92 amino acid fragment of T24 isimmunoreactive with T. solium T24 antibodies with at least 95%sensitivity and at least 92% specificity.

EXAMPLE 4 Stable Expression of T24H

This example describes methods that were used to generate a stable cellline expressing recombinant T24H.

The recombinant vector, pMT/BiP/T24H (see Example 3), and the selectionplasmid, pCoBlast (Invitrogen) which contains an ampicillin and ablasticidin gene, were co-transfected into S2 cells using thecalcium-phosphate method. Blasticidin was added to the culture medium 96hours post-transfection and the selective medium was maintained for 18days. For expression, the stable cell line was induced with coppersulfate and the supernatant harvested at 96 hours. Western blot stripswere made and tested with the same sera used to test the transientlyexpressed T24H described in Example 3. The results obtained using stablyexpressed T24H were the same as obtained with transiently expressedT24H.

EXAMPLE 5 Recombinant Expression in Insect Cells

Examples 2-4 describe expression of T24 and T24H in S2 insect cells,according to the manufacturer's instructions (Invitrogen). However, oneskilled in the art will appreciate that other insect cells can be used.This example describes methods that can be used to express T24 andfragments thereof recombinantly in other insect cell lines.

The coding region of SEQ ID NO: 1, a fragment of SEQ ID NO: 1 (such asnucleotides 33-710 or 342-617), or variants of SEQ ID NO: 1 can besubcloned into the desired vector using standard molecular biologytechniques.

For example, expression of T24 or T24H behind the honeybee melittinsecretory signal or behind the BiP secretory signal can be performed invarious insect cell lines, to allow secretion of the recombinant proteininto the culture medium. The vector pMIB/V5-His A (Invitrogen) can beused to constitutively express T24 or T24H behind the honeybee melittinsecretion signal in lepidopteran cells (such as Sf9, Sf21, High Fivecells, all available from Invitrogen). Methods for generating therecombinant plasmids, transfecting them into insect cells (for exampleusing lipid-mediated transfection), expressing, and purifying therecombinant protein are known in the art. In addition, instructions areprovided by Invitrogen.

The vector pIZ/V5-His (Invitrogen) with BiP/T24H cloned into it can beused to constitutively express T24 or T24H behind the BiP secretionsignal in any lepidopteran cell such as Sf9 and High Five cells. Thesequence for BiP is from the vector pMT/BiP/V5-His. Methods forgenerating the recombinant plasmids, transfecting them into insect cells(for example using lipid-mediated transfection), expressing, andpurifying the recombinant protein are known in the art. In addition,instructions are provided by Invitrogen.

A baculovirus transfer vector can also be used to produce recombinantT24 protein. Recombinant virus containing a T24 or T24H is generated byco-transfection of the transfer vector with Bac-N-Blue AcMNPV linearDNA, a modified baculovirus vector, in insect cells such as Sf9 cells.After purification of the recombinant virus, cells are infected andharvested at 96 hours post-infection as described above.

Total cell lysates from cultures transfected or infected with arecombinant T24 or T24H sequence can be analyzed by immunoblot asdescribed in the above examples.

Other insect cells that can be used include, but are not limited to, D.melanogaster Kc1 cells and gypsy moth cell line, IPLB-LdEIta (Ld).

EXAMPLE 6 Method for Detecting the Presence of T24 Antibodies in aSample

This example describes methods that can be used to determine if a samplecontains T24 antibodies. In some examples, the presence of T24antibodies indicates that the subject has cysticercosis.

Recombinant or synthetic T24 proteins, such as those described in theexamples above are purified to at least 50% purity, for example byresolving on SDS-PAGE, blotted onto a substrate (such as nitrocelluloseor PVDF), and probed with a sample (such as sera) obtained from asubject having or suspected of having cysticercosis. The sample can beany biological sample, such as sera, saliva, or urine, or any sample inwhich T24 antibodies reside. If anti-cysticercosis T24 antibodies arepresent in the sample, they will specifically recognize and bind to theT24 proteins present in the substrate, thereby forming antibody-proteincomplexes. As noted above, full-length recombinant T24 migrates inSDS-PAGE at a little less than 30 kDa, while T24H migrates at about 10kDa.

Detection of antibody-protein complexes can be achieved by labeling theantibody, protein, or both, with a detectable label. For example,following formation of antibody-protein complexes, the substratecontaining the T24 protein can be probed with a secondary antibodycontaining a detectable label (such as a fluorophore). The secondaryantibody can recognize the T24 protein or the anti-T24 antibody presentin the biological sample. The detection of an interaction between arecombinant T24 protein and anti-T24 antibodies in the sample indicatesthat the subject has or had cysticercosis.

Ideally, little to no detectable reactivity with recombinant T24 isobserved with a control sample from normal human sera (a subject notinfected by T. solium) or a sample infected with another pathogen, suchas Echinococcus.

EXAMPLE 7 Method for Detecting the Presence of T24 Sequences in a Sample

This example describes methods that can be used to determine if a samplecontains T24 proteins or nucleic acid molecules. The disclosure of T24protein and nucleic acid sequences allows those skilled in the art touse molecular biology methods to identify the presence of such sequencesin a sample. In some examples, the presence of T24 proteins or nucleicacid molecules indicates that the subject has cysticercosis.

For example, primers can be generated based on SEQ ID NO: 1 that allowone to detect T24 sequences in a sample. In one example, the primers areused to amplify T24 sequences present in a sample, for example by usingPCR or real time PCR.

In another example, probes containing a detectable label are generatedbased on SEQ ID NO: 1. Probes are contacted with a sample for a timesufficient to allow hybridization between the probe and a T24 nucleicacid sequence present in the sample. Resulting probe:T24 nucleic acidsequence complexes can then be detected. In some examples, the probe isconjugated to a solid substrate, such as a bead. In particular examples,nucleic acid molecules are isolated from a sample prior to exposing thenucleic acid sequences to a probe, for example by running the sample ona gel (such as an agarose or acrylamide gel). The gel is then contactedwith a substrate such as nitrocellulose, to allow transfer of thenucleic acid sequences to the substrate. The substrate is contacted withthe probe or a time sufficient to allow hybridization between the probeand a T24 nucleic acid sequence present in the substrate. Resultingprobe:T24 nucleic acid sequence complexes can then be detected.

To detect T24 proteins present in a sample, antibodies that recognizeT24 can be used (see Example 8). For example, proteins present in asample can be purified, for example by running on an SDS-PAG, andtransferred to a substrate. The substrate is then probed with anantibody that recognizes T24, thereby generating T24 protein-antibodycomplexes which are detected. The antibody can include a detectablelabel.

EXAMPLE 8 Production and Use of Antibodies

Monoclonal or polyclonal antibodies can be produced to a T24 protein,such as SEQ ID NO: 2, or variants, fragments, and fusions thereof (suchas amino acids 104-195 of SEQ ID NO: 2). Optimally, antibodies raisedagainst a T24 protein will specifically detect the protein. That is,antibodies raised against a T24 protein recognize and bind T24 proteinbut do not substantially recognize or bind to other proteins found inhuman or pig cells. The determination that an antibody specificallydetects an protein is made using any standard immunoassay methods; forinstance, Western blotting (Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). Additionally, chimeric antibodies can be produced (for example,Morrison et al., J. Bacteriol. 159:870, 1984; Neuberger et al., Nature312:604-8, 1984; and Takeda et al., Nature 314:4524, 1985), as well assingle-chain antibodies (for example, U.S. Pat. Nos: 5,476,786;5,132,405; and 4,946,778).

The T24 protein can be obtained and purified from T. solium.Alternatively, T24 protein can be recombinantly generated (see Examples2-5) or synthetically produced now that protein and nucleic acidsequences are known.

To determine that an antibody preparation (such as one produced in amouse against a T24 protein) specifically detects the T24 protein byWestern blotting, total cellular protein is extracted from cells (suchas human or pig cells) and electrophoresed on a sodium dodecylsulfate-polyacrylamide gel. The proteins are transferred to a membrane(for example, nitrocellulose) and the antibody preparation incubatedwith the membrane. After washing the membrane to remove non-specificallybound antibodies, the presence of specifically bound antibodies isdetected by using an appropriate secondary antibody (such as ananti-mouse antibody or an anti-rabbit antibody) conjugated to a label(for example an enzyme such as alkaline phosphatase where application ofsubstrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazoliumresults in the production of a dense blue compound by immuno-localizedalkaline phosphatase).

Antibodies that specifically detect a T24 protein will, by thistechnique, be shown to bind to the protein band (such as the T24 band,which localizes at a given position on the gel determined by itsmolecular weight and phosphorylation; see Examples above). Non-specificbinding of the antibody to other proteins may occur and may bedetectable as a weak signal on the Western blot The non-specific natureof this binding is recognized by one skilled in the art by the weaksignal obtained on the Western blot relative to the strong primarysignal arising from the specific antibody-T24 protein binding.

Substantially pure T24 proteins suitable for use as an immunogen isisolated as herein described, for example at least 50% pure by weight,for example at least 75%, at least 90%, or even at least 98% pure.Concentration of protein in the final preparation is adjusted, forexample, by concentration on an Anmicon filter device, to the level of afew μg/ml. Monoclonal or polyclonal antibody to the protein can then beprepared.

Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of a T24 protein can be identified,isolated and prepared from murine hybridomas using the method of Kohlerand Milstein (Nature 256:495, 1975), using the human B-cell method(Kosbor et al., Immunology Today 4:72, 1983), or the EBV-hybridomamethod (Cole et al., in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96, 1983), or derivative thereof.

For example, a mouse is repetitively inoculated with a few μg of a T24protein over a period of a few weeks. The mouse is sacrificed andantibody-producing cells of the spleen isolated. The spleen cells arefused using polyethylene glycol with mouse myeloma cells, and the excessunfused cells destroyed by growth of the system on selective mediacomprising aminopterin (HAT media). The fused cells are diluted andaliquots of the dilution placed in wells of a microtiter plate wheregrowth of the culture is continued.

Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as described by Engvall (Enzymol. 70:419, 1980), and similar methods.Selected positive clones can be expanded and their monoclonal antibodyproduct harvested for use. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane (Antibodies: A LaboratoryManual. 1988, Cold Spring Harbor Laboratory, New York). In addition,protocols for producing humanized forms of monoclonal antibodies (fortherapeutic applications) and fragments of monoclonal antibodies areknown in the art.

Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes ofT24 protein can be prepared by immunizing suitable animals with theexpressed protein, which can be unmodified or modified to enhanceimmunogenicity. Effective polyclonal antibody production is affected bymany factors related to both the antigen and the host species. Forexample, small molecules tend to be less immunogenic than others and mayrequire the use of carriers and adjuvant. Also, host animals vary inresponse to site of inoculations and dose, with both inadequate orexcessive doses of antigen resulting in low titer antisera. Small doses(ng level) of antigen administered at multiple intradermal sites appearsto be most reliable. An effective immunization protocol for rabbits canbe found in Vaitukaitis et al. (J. Clin. Endocrinol. Metab. 33:988-91,1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al. (In: Handbook of Experimental Immunology,Wier, D. (ed.). Chapter 19. Blackwell. 1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher (Manualof Clinical Immunology, Chapter 42. 1980).

Antibodies Raised by Injection of a cDNA

Antibodies can be raised against a T24 protein by subcutaneous injectionof a DNA vector expressing the protein into an animal, such as mice orrabbits. Delivery of the recombinant vector into the animal can beachieved using a hand-held form of the Biolistic system (Sanford et al.,Particulate Sci. Technol. 5:27-37, 1987) described by Tang et al.(Nature 356:152-4, 1992). Expression vectors include recombinant vectorsexpressing a cDNA under transcriptional control of the human β-actinpromoter or cytomegalovirus (CMV) promoter.

Labeled Antibodies

T24 antibodies can be conjugated with a label for their direct detection(see Chapter 9, Harlow and Lane, Antibodies: A Laboratory Manual. 1988).Exemplary labels include radiolabels, enzymes (such as alkalinephosphatase (AP) or HRP), colorimetric labels, chelating agents,fluorescent labels, colloidal gold, ligands (such as biotin), andchemiluminescent agents, and is chosen based on the method of detectionavailable to the user. The method of producing these conjugates isdetermined by the reactive group on the label added.

For example, an antibody can be radiolabeled with iodine (¹²⁵I), whichyields low-energy gamma and X-ray radiation. Briefly, 10 μg of proteinin 25 μl of 0.5 M sodium phosphate (pH 7.50 is placed in a 1.5 mlconical tube. To this, 500 μC of Na¹²⁵I, and ²⁵ μl of 2 mg/ml chloramineT is added and incubated for 60 seconds at RT. To stop the reaction, 50μl of chloramine T stop buffer is added (2.4 mg/ml sodium metabisulfite,10 mg/ml tyrosine, 10% glycerol, 0.1% xylene cyanol in PBS). Theiodinated antibody is separated from the iodotyrosine on a gelfiltration column.

Uses for T24 Antibodies

T24 antibodies prepared according to these methods can be used in anassay to determine the presence of T24 or T. solium in a sample, such asa quantitative or or semi-quantitative assay.

EXAMPLE 9 Antigenic Compositions

This example describes compositions that include T24 proteins (as wellas fragments, fusions, and variants thereof) that can be used tostimulate an immune response in a subject. Stimulation of an immuneresponse against a T24 protein, for example in a pig or human subject,can protect a subject against T. solium infection, cysticercosis,neurocysticercosis, or combinations thereof.

T24 recombinant or synthetic proteins can be present alone or with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers include agents that aid in stimulation of the immune response,for example an adjuvant. Adjuvants are nonspecific immune stimulatorsthat increase the immune readiness and aid in stimulating a higher level(titer) of serum antibodies that recognize the epitopic peptidesequences. Adjuvants include, for example, Freund's complete adjuvant,Freund's incomplete adjuvant, B30-MDP, LA-15-PH, montanide, saponin,aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, amycobacterial antigen, and combinations thereof. If the adjuvant is alipid it may be linked to the epitopic peptide(s). A high titer ofantibodies serves to protect a subject from the pathogen to which theantibodies are directed.

Other examples of pharmaceutically acceptable carriers includephysiologically acceptable masses to which the T24 eptiope it attached,and in some examples, enhances the immune response. In one example, amass is one or more amino acids or other moieties, such as a dimer,oligomer, or higher molecular weight polymer of a sequence of aminoacids of a T24 epitope. In other words, a T24 epitope can be formed fromnaturally available materials or synthetically produced and can then bepolymerized to build a chain of two or more repeating units so that therepeating sequences form both the carrier and the immunogenicpolypeptide. Alternatively, additional amino acids can be added to oneor both ends of a T24 peptide. Polysaccharides can also attached to thedisclosed epitopes, and include those of molecular weight 10,000 to1,000,000, such as starches, dextran, agarose, ficoll, or its carboxylmethyl derivative and carboxy methyl cellulose. Polyamino acids can alsoattached to the disclosed epitopes, and include, polylysine, polyalanylpolylysine, polyglutamic acid, polyaspartic acid and poly (C₂-C₁₀) aminoacids.

Organic polymers can also attached to the disclosed T24 epitopes.Examples of such polymers include, but are not limited to, polymers andcopolymers of amines, amides, olefins, vinyls, esters, acetals,polyamides, carbonates and ethers and the like. Generally, the molecularweight of these polymers will vary dramatically. The polymers can havefrom two repeating units up to several thousand, for example twothousand repeating units. The number of repeating units will beconsistent with the use of the immunizing composition in a host animal.Generally, such polymers have a lower molecular weight, for example,between 10,000 and 100,000 kD (the molecular weight being determined byultracentrifugation). Inorganic polymers can also be employed. Theseinorganic polymers can be inorganic polymers containing organicmoieties. In particular, silicates and aluminum hydroxide can be used ascarriers. Ideally, the carrier is an immunological adjuvant, such asmuramyl dipeptide or its analogs.

Methods of administering the disclosed compositions to a subject areprovided in Example 10.

Regardless of the exact formulation, T24 peptides (and nucleic acidsencoding such epitopes), such as SEQ ID NOS: 1 and 2, or variants,fragments, fusions, and mixtures thereof, can be tested for theirpotential as an immunogenic molecule(s) or compositions with bindingassays, in vitro cell culture techniques and in small mammal models.Proliferative assays can be used to measure the ability of T24 peptidesto stimulate a T-cell response (PCT publication WO 02/22860). T-cells(2×104) or irradiated peripheral blood mononuclear cells (5×10⁴) areseeded, in duplicate, into wells with or without about 200 μg/ml peptide(Hemmer, et al., 1998, J. Pept. Res. 52:33845). Proliferation ismeasured by ³H-thymidine incorporation (Hemmer et al., 1997, Exp. Med.185:1651-9).

The T24 peptides disclosed herein can also be tested in a cytotoxic Tlymphocyte (CTL) assay (see, for example, Sette et al., 1994. J.Immunol. 153:5586-92, and PCT publication WO 01/55177). Briefly, thespleen of peptide immunized transgenic mice are collected aseptically 10days after immunization and placed in 5 ml of cell medium (RMPI 1640,penicillin +streptomycin, 2% Hepes buffer, 10% Fetal calf serum) on ice.The splenocytes are cultured for 6 days in the presence of LPS blastscoated with 100 tig/ml of the peptide (stimulator cells) and thenassayed for peptide-specific CTL activity by using EL4-A2 and EL4 celllines in the presence or absence of the query peptides.

EXAMPLE 10 Administration of Therapeutic Agents

Administering a T24 peptide, nucleic acid molecule, or antibody of thepresent disclosure can be accomplished by any means known to the skilledartisan. For example, a pharmaceutically acceptable carrier can beprovided for a T24 peptide, nucleic acid molecule, or antibody. Examplesof pharmaceutically acceptable carriers include, but are not limited to,substances that are animal, vegetable, or mineral in origin, those thatare physiologically acceptable and function to present a T24 epitope tothe immune system. Thus, a wide variety of pharmaceutically acceptablecarriers are acceptable, and include materials which are inert, or whichhave biological activity or promote an immune response.

Pharmaceutical preparations can contain only one type of therapeuticmolecule, or can include a combination of several types of therapeuticmolecules, such as other anti-cysticercosis or anti-helminthic agents,for example oxfendazole, albendazole, or praziquantel. In general, thenature of the pharmaceutically acceptable carrier will depend on theparticular mode of administration being employed.

The pharmaceutical compositions disclosed herein can be prepared andadministered in dose units. Solid dose units include tablets, capsules,transdermal delivery systems, and suppositories. For treatment of asubject, depending on activity of the compound, manner ofadministration, nature and severity of the disorder, age and body weightof the subject, different daily doses are necessary. Under certaincircumstances, however, higher or lower daily doses may be appropriate.The administration of a therapeutic amount can be carried out both bysingle administration in the form of an individual dose unit or elseseveral smaller dose units and also by multiple administration ofsubdivided doses at specific intervals.

The T24 peptides, nucleic acids, and antibodies and pharmaceuticalcompositions disclosed herein can be administered by any method used inthe art, for example locally or systemically, such as topically,intravenously, orally, parenterally or as implants. Suitable solid orliquid pharmaceutical preparation forms are, for example, granules,powders, tablets, coated tablets, (micro)capsules, suppositories,syrups, emulsions, suspensions, creams, aerosols, drops or injectablesolution in ampule form and also preparations with protracted release ofactive compounds, in whose preparation excipients and additives orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of present methods for drug delivery, see Langer, Science249:1527-33, 1990 (incorporated herein by reference).

In one example, T24 peptides, nucleic acids, and antibodies areadministered to a subject in the presence of a lipid of lipoprotein.Liposomes are artificial membrane vesicles that are useful as deliveryvehicles in vitro and in vivo. In one example, a liposome includes thedesired molecule and is directed to the site of lymphoid cells, wherethe liposomes then deliver the selected therapeutic molecule. Largeuni-lamellar vesicles (LUV), which range in size from 0.2-4.0 μm, canencapsulate a substantial percentage of an aqueous buffer containinglarge macromolecules. Nucleic acid molecules, proteins, and antibodiescan be encapsulated within the aqueous interior and be delivered tocells in a biologically active form (Fraley et al., 1981, TrendsBiochem. Sci. 6:77, 1981).

Liposomes can be formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids (such ashigh-phase-transition-temperature phospholipids) in combination with asterol, such as cholesterol. However, other lipids can be used, such asphosphatidyl compounds, for example phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Other lipids that can beused include diacylphosphatidyl-glycerols, where the lipid moietycontains 14-18 carbon atoms, such as 16-18 carbon atoms, and issaturated. Additional phospholipids that can be used include, but arenot limited to, egg phosphatidylcholine, dipalmitoylphosphatidylcholineand distearoylphosphatidylcholine.

The selection of lipids is generally guided by consideration of, forexample, liposome size and stability of the liposomes in the bloodstream. A variety of methods are available for preparing liposomes andare described, for example, in Szoka et al., 1980, Ann. Rev. Biophys.Bioeng 9:467 and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369. Particular lipid residues, such as palmitic acid or otheruncharged fatty acid residues of different chain lengths and degrees ofunsaturation, ranging from acetic to stearic acid as well as tonegatively charged succinyl residues can be attached to the epitopic viathe appropriate carboxylic acid anhydrides.

The lipids can be directly attached to a T24 epitopic peptide orindirectly through a linkage as described above. For example, a lipidcan be attached directly to the amino terminus of the peptide or via alinkage such as Ser-Ser, Gly, Gly-Gly, or Ser.

The surface of a targeted delivery system can be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

For administration of T24 nucleic acid molecules, vectors can be used.In one example, the vector is a viral vector, such as an adenovirus,herpes virus, vaccinia, or an RNA virus such as a retrovirus. In oneexample, the retroviral vector is a derivative of a murine or avianretrovirus. Examples of retroviral vectors in which a single foreigngene can be inserted include, but are not limited to: Moloney murineleukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murinemammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). When thesubject is a human, a vector such as the gibbon ape leukemia virus(GaLV) can be utilized. A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. By inserting a nucleic acid sequenceencoding a T24 peptide into the viral vector, along with another genethat encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by attaching, for example, a sugar, a glycolipid,or a protein. Targeting can be accomplished by using an antibody totarget the retroviral vector. Those of skill in the art will know of, orcan readily ascertain without undue experimentation, specificpolynucleotide sequences that can be inserted into the retroviral genomeor attached to a viral envelope to allow target specific delivery of theretroviral vector containing a nucleotide sequence encoding a T24epitope.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence that enables the packaging mechanism to recognize anRNA transcript for encapsidation. Helper cell lines that have deletionsof the packaging signal include, but are not limited to Q2, PA317, andPA12, for example. These cell lines produce empty virions, since nogenome is packaged. If a retroviral vector is introduced into such cellsin which the packaging signal is intact, but the structural genes arereplaced by other genes of interest, the vector can be packaged andvector virion produced.

Another targeted delivery system for the therapeutic polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes.

Amounts effective for therapeutic use can depend on the severity of thedisease and the age, weight, general state of the patient, and otherclinical factors. Thus, the final determination of the appropriatetreatment regimen will be made by the attending clinician. Dosages usedin vitro can provide useful guidance in the amounts useful for in situadministration of the pharmaceutical composition, and animal models canbe used to determine effective dosages for treatment of particulardisorders. Various considerations are described, for example in Gilmanet al., eds., Goodman and Gilman: The Pharmacological Bases ofTherapeutics, 8th ed., Pergamon Press, 1990; and Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Co., Easton, Pa.,1990.

Typically, the dose range for a T24 protein is from about 0.1 μg/kg bodyweight to about 100 mg/kg body weight. Other suitable ranges includedoses of from about 1 μg/kg to 10 mg/kg body weight. In one example, thedose is about 1.0 μg to about 50 mg, for example, 1 μg to 1 mg, such as1 mg peptide per subject. The dosing schedule can vary from daily to asseldom as once a year, depending on clinical factors, such as thesubject's sensitivity to the peptide and tempo of their disease.Therefore, a subject can receive a first dose of immunogenic T24peptide, and then receive a second dose (or even more doses>at somelater time(s), such as at least one day later, such as at least one weeklater, such as at least one month later. In one example, initialimmunization can be followed by boosting dosages of from about 1 μg to50 mg, for example, 1 μg to 500 μg, such as 1 μg to about 250 μg ofpeptide. A boosting regimen can be followed over weeks to months,depending upon the patient's response and condition by measuringspecific immune activity in the patient's blood. In the case of a moreaggressive disease it can be preferable to administer doses such asthose described above by alternate routes including intravenously orintrathecally. Continuous infusion may also be appropriate.

Protein-based pharmaceuticals may be inefficiently delivered throughingestion. However, pill-based forms of pharmaceutical proteins can beadministered subcutaneously, particularly if formulated in aslow-release composition. Slow-release formulations can be produced bycombining the target protein with a biocompatible matrix, such ascholesterol. Another possible method of administering proteinpharmaceuticals is through the use of mini osmotic pumps. As statedabove a biocompatible carrier would also be used in conjunction withthis method of delivery.

The disclosed peptides can also be used as diagnostic reagents. Forexample, a T24 immunogenic peptide can be used to determine thesusceptibility of a particular individual to a treatment regimen thatemploys the peptide or related peptides. As diagnostic reagents,epitopic peptides can be helpful in modifying an existing treatmentprotocol or in determining a prognosis for an affected individual.

In view of the many possible embodiments to which the principles of ourdisclosure may be applied, it should be recognized that the illustratedembodiments are only particular examples of the disclosure and shouldnot be taken as a limitation on the scope of the disclosure. Rather, thescope of the disclosure is in accord with the following claims. Wetherefore claim as our invention all that comes within the scope andspirit of these claims.

1. A purified peptide comprising at least 20 contiguous amino acids fromamino acids 104-195 of SEQ ID NO: 2, wherein the purified peptide isimmunogenic, and wherein the purified peptide has a purity of at least60% by weight.
 2. The purified peptide of claim 1, wherein the sequenceis at least 90% identical to amino acids 104-195 of SEQ ID NO:
 2. 3. Thepurified peptide of claim 1, wherein the sequence is at least 95%identical to amino acids 104-195 of SEQ ID NO:
 2. 4. The purifiedpeptide of claim 1, wherein the amino acid sequence comprises aminoacids 104-195 of SEQ ID NO:
 2. 5. The purified peptide of claim 1,wherein the purified peptide consists of amino acids 104-195 of SEQ IDNO:
 2. 6. The purified peptide of claim 1, wherein the sequence is atleast 90% identical to SEQ ID NO:
 2. 7. The purified peptide of claim 1,wherein the sequence is at least 95% identical to SEQ ID NO:
 2. 8. Thepurified peptide of claim 1, wherein the amino acid sequence comprisesSEQ ID NO:
 2. 9. The purified peptide of claim 1, wherein the purifiedpeptide consists of SEQ ID NO:
 2. 10. The purified peptide of claim 1,wherein the purified peptide comprises no more than two amino acidsubstitutions within amino acids 104-195 of SEQ ID NO:
 2. 11. Thepurified peptide of claim 1, wherein the purified peptide comprisestwo-ten amino acid conservative substitutions within amino acids 104-195of SEQ ID NO:
 2. 12. The purified peptide of claim 1, wherein thepeptide is a recombinant or synthetic peptide.
 13. The purified peptideof claim 1, wherein the peptide is at least 50% pure.
 14. An isolatednucleic acid encoding the purified peptide of claim
 1. 15. The isolatednucleic acid of claim 14, wherein the nucleic acid sequence comprises atleast 90% identity to nucleotides 342-617 of SEQ ID NO:
 1. 16. Theisolated nucleic acid of claim 14, wherein the nucleic acid sequencecomprises at least 95% identity to nucleotides 342-617 of SEQ ID NO: 1.17. The isolated nucleic acid of claim 14, wherein the nucleic acidsequence comprises nucleotides 342-617 of SEQ ID NO:
 1. 18. The isolatednucleic acid of claim 14, wherein the nucleic acid sequence comprises atleast 90% identity to nucleotides 33-707 of SEQ ID NO:
 1. 19. Theisolated nucleic acid of claim 14, wherein the nucleic acid sequencecomprises at least 95% identity to nucleotides 33-707 of SEQ ID NO: 1.20. The isolated nucleic acid of claim 14, wherein the nucleic acidsequence comprises nucleotides 33-707 of SEQ ID NO:
 1. 21. The isolatednucleic acid of claim 14, wherein the nucleic acid sequence is operablylinked to a promoter.
 22. A vector comprising the nucleic acid of claim14.
 23. The vector of claim 22, wherein the vector is a viral vector.24. The vector of claim 22, wherein the vector is a plasmid vector. 25.A host cell transformed with the vector of claim
 22. 26. The host cellof claim 25, wherein the host cell is an insect cell.
 27. A compositioncomprising the purified peptide of claim 1 or the isolated nucleic acidof claim
 14. 28. The composition of claim 27, wherein the purifiedpeptide of claim 1 is a recombinant or synthetic peptide.
 29. Thecomposition of claim 27, further comprising an adjuvant.
 30. Thecomposition of claim 27, further comprising a pharmaceuticallyacceptable carrier.
 31. A composition for detecting the presence of T.solium in a sample, comprising a nucleic acid probe comprising 30 ormore contiguous nucleotides capable of hybridizing under stringentconditions with SEQ ID NO:
 1. 32. A composition for detecting thepresence of T. solium in a sample, comprising the purified peptide ofclaim
 1. 33. A method of detecting a presence of T. solium in a sample,comprising: contacting the composition of claim 31 with the sample; anddetecting hybridization of the nucleic acid probe to the sample, whereindetection of the hybridization indicates the presence of T. solium inthe sample.
 34. The method of claim 33, wherein the nucleic acid probecomprises a detectable label, and wherein detection of the detectablelabel indicates the presence of T. solium in the sample.
 35. A methodfor detecting T. solium antibodies in a sample comprising: contactingthe sample with the composition of claim 28; and detecting the formationof a complex between the recombinant or synthetic peptide and a T24antibody in the sample, wherein a presence of an antibody—peptidecomplex indicates the presence of T. solium antibodies in the sample.36. The method of claim 35, wherein the recombinant or synthetic peptidecomprises at least 90% sequence identity to amino acids 104-195 of SEQID NO:
 2. 37. The method of claim 35, wherein the method furthercomprises contacting the sample with one or more of gp50, gp42, gp21,gp18, gp14, or gp13 larval T. solium peptides.
 38. The method of claim35 wherein the peptide is encoded by a nucleic acid molecule comprisingnucleotides 342-617 of SEQ ID NO:
 1. 39. The method of claim 35, whereinthe method is a method for diagnosing a T solium associated disease orcondition in a mammal, and wherein the presence of T. solium antibodiesin the sample indicates the presence of the T. solium associated diseaseor condition in the mammal.
 40. The method of claim 39, wherein the T.solium associated disease or condition is cystercercosis orneurocystercercosis.
 41. A method of eliciting an immune response in asubject, comprising administering to the subject a first dose of atherapeutically effective amount of the peptide of claim 1, whereinadministration of the peptide to the subject results in elicitation ofthe immune response against T24.
 42. The method of claim 41, wherein thesubject has cysticercosis or neurocysticercosis.
 43. The method of claim41, wherein a susceptibility of the subject to cysticercosis orneurocysticercosis is determined prior to administering to the subject atherapeutically effective amount of the peptide.
 44. The method of claim41, further comprising administering a second dose of a therapeuticallyeffective amount of the purified peptide of claim 1, at a time after thefirst dose.
 45. A method of treating cysticercosis or neurocysticercosisin a subject, comprising administering to the subject a therapeuticallyeffective amount of the peptide of claim 1, thereby treating thecysticercosis or neurocysticercosis in the subject.
 46. An isolatedspecific binding agent that recognizes the peptide of claim
 1. 47. Theisolated specific binding agent of claim 46, wherein the specificbinding agent is an antibody.
 48. A method of generating antibodiesspecific for a T24 peptide, comprising introducing into a subject thepurified peptide of claim
 1. 49. The method of claim 38, wherein thesubject is an experimental animal.
 50. The method of claim 45, whereinthe method comprises administering to the subject a therapeuticallyeffecting amount of a peptide comprising at least 90% sequence identityto amino acids 104-195 of SEQ ID NO: 2.